Artificial T helper cell epitopes as immune stimulators for synthetic peptide immunogens

ABSTRACT

The present invention is directed to novel artificial T helper cell epitopes (Th epitopes) designed to provide optimum immunogenicity when used in peptide immunogens comprising B cell epitopes or peptide haptens, a target antigenic site of a target antigen for eliciting antibodies thereto. The artificial Th epitopes are covalently linked to the target antigenic site and and optionally an immunostimulatory sequence to provide effective and safe peptide immunogens.

This is a Continuation-In-Part of application Ser. No. 09/100,412, filedJun. 20, 1998, now abandon.

FIELD OF THE INVENTION

This invention relates to a peptide immunogen comprising a novelartificial T helper cell epitope (Th) covalently linked to a desiredtarget antigenic site comprising B cell epitopes and optionally ageneral immune stimulator sequence. The artificial Th epitope imparts tothe peptide immunogen the capability to induce strong T helpercell-mediated immune responses and the production of antibodies directedagainst the “target antigenic site.” The invention also provides for theadvantageous replacement of carrier proteins and pathogen-derived Thelper cell sites in established peptide immunogens by the novelartificial T helper cell epitopes for improved immunogenicity.

Many rules have been developed for predicting the amino acid sequencesof T cell epitopes. However, because there is no central unifying theoryon how or what makes a particular amino acid sequence useful as a T cellepitope, the rules are empirical and are not universally applicable.Being aware of these rules, the novel artificial T helper cell epitopesof the present invention were developed, nevertheless, by empiricalresearch.

The peptide immunogens of the present invention are useful for evokingantibody responses in an immunized host to a desired target antigenicsite, including sites taken from pathogenic organisms, and sites takenfrom normally immunosilent self-antigens and tumor-associated targets.Accordingly, the peptides of the invention are useful in diverse medicaland veterinary applications, such as: vaccines to provide protectiveimmunity from infectious disease; immunotherapies for treating disordersresulting from malfunctioning normal physiological processes;immunotherapies for treating cancer and as agents to intervene in normalphysiological processes to produce desirable results.

For example, the novel artificial T helper cell epitopes of the presentinvention provide novel short peptide immunogens that elicit antibodiestargeted to luteinizing hormone-releasing hormone (LHRH) and are usefulfor contraception, control of hormone-dependent tumors, prevention ofboar taint, and immunocastration. The novel artificial Th epitopes ofthe present invention have been found to provoke an immune response whencombined with target B cell epitopes of variousmicroorganisms/proteins/peptides. In addition to LHRH, the artificial Thepitopes of the present invention have been found to be useful whenlinked to other target antigenic sites including somatostatin for growthpromotion in farm animals; IgE for treatment of allergy; the CD4receptor of T helper cells for treatment and prevention of HIV infectionand immune disorders; foot-and-mouth disease virus capsid protein forprevention of foot-and-mouth disease; HIV virion epitopes for preventionand treatment of HIV infection; the circumsporozoite antigen ofPlasmodium falciparum for prevention and treatment of malaria; andCholesteryl ester transport protein (CETP) for prevention and treatmentof arteriosclerosis.

BACKGROUND OF THE INVENTION

It is known that most antibody immune responses are cell-mediated,requiring cooperative interaction between antigen-presenting cells, Bcells (antibody-producing cells which also function asantigen-presenting cells), and T helper (Th) cells. Consequently, theelicitation of an effective antibody response requires that the B cellsrecognize the target antigenic site (B cell epitope) of a subjectimmunogen and the T helper cells recognize a Th epitope. Generally, theT helper epitope on a subject immunogen is different from its B cellepitope(s) (Babbitt et al., Nature, 1985; 317: 359-361). The B cellepitope is a site on the desired target recognized by B cells which inresponse produce antibodies to the desired target site. It is understoodthat the natural conformation of the target determines the site to whichthe antibody directly binds. The T helper cell recognition of proteinsis, however, much more complex and less well understood. (Cornette etal., in Methods in Enzymology, vol 178, Academic Press, 1989, pp611-634).

Under present theories, evocation of a Th cell response requires the Thelper cell receptor to recognize not the desired target but a complexon the membrane of the antigen-presenting cell formed between aprocessed peptide fragment of the target protein and an associated classII major histocompatibility complex (MHC). Thus, peptide processing ofthe target protein and a three-way recognition is required for the Thelper cell response. The three part complex is particularly difficultto define since the critical MHC class II contact residues are variablypositioned within different MHC binding peptides (Th epitopes) and thesepeptides are of variable lengths with different amino acid sequences(Rudensky et al., Nature, 1991; 353:622-627). Furthermore, the MHC classII molecules themselves are highly diverse depending on the geneticmake-up of the host. The immune responsiveness to a particular Thepitope is thus in part determined by the MHC genes of the host. Infact, it has been shown that certain peptides only bind to the productsof particular class II MHC alleles. Thus, it is difficult to identifypromiscuous Th epitopes, i.e., those that are reactive across speciesand across individuals of a single species. It has been found that thereactivity of Th epitopes is different even among individuals of apopulation.

The multiple and varied factors for each of the component steps of Tcell recognition: the appropriate peptide processing by theantigen-processing cell, the presentation of the peptide by agenetically determined class II MHC molecule, and the recognition of theMHC molecule/peptide complex by the receptor on T helper cells have madeit difficult to determine the requirements for promiscuous Th epitopesthat provide for broad responsiveness (Bianchi et al., EP 0427347;Sinigaglia et al., chapter 6 in Immunological Recognition of Peptides inMedicine and Biology, ed., Zegers et al., CRC Press, 1995, pp 79-87).

It is clear that for the induction of antibodies, the immunogen mustcomprise both the B cell determinant and Th cell determinant(s).Commonly, to increase the immunogenicity of a target, the Th response isprovided by coupling the target to a carrier protein. The disadvantagesof this technique are many. It is difficult to manufacture well-defined,safe, and effective peptide-carrier protein conjugates for the followingreasons:

1. Chemical coupling are random reactions introducing heterogeneity ofsize and composition, e.g., conjugation with glutataraldehyde(Borras-Cuesta et al., Eur J Immunol, 1987; 17: 1213-1215);

2. the carrier protein introduces a potential for undesirable immuneresponses such as allergic and autoimmune reactions (Bixler et al., WO89/06974);

3. the large peptide-carrier protein elicits irrelevant immune responsespredominantly misdirected to the carrier protein rather than the targetsite (Cease et al., Proc Natl Acad Sci USA, 1987; 84: 4249-4253); and

4. the carrier protein also introduces a potential for epitopicsuppression in a host which had previously been immunized with animmunogen comprising the same carrier protein. When a host issubsequently immunized with another immunogen wherein the same carrierprotein is coupled to a different hapten, the resultant immune responseis enhanced for the carrier protein but inhibited for the hapten(Schutze et al., J Immunol, 1985; 135: 2319-2322).

To avoid these risks, it is desirable to replace the carrier proteins. Tcell help may be supplied to a target antigen peptide by covalentbinding to a well-characterized promiscuous Th determinant. Knownpromiscuous Th are derived from the potent pathogenic agents such asmeasles virus F protein (Greenstein et al., J Immunol, 1992; 148:3970-3977) and hepatitis B virus surface antigen (Partidos et al., J GenVirol 1991; 72: 1293-1299). The present inventors have shown that manyof the known promiscuous Th are effective in potentiating a poorlyimmunogenic peptide, such as the decapeptide hormone luteinizinghormone-releasing hormone (LHRH) (U.S. Pat. No. 5,759,551). Otherchimeric peptides comprising known promiscuous Th epitopes with poorlyimmunogenic synthetic peptides to generate potent immunogens have beendeveloped (Borras-Cuesta et al., 1987). Well-designed promiscuous Th/Bcell epitope chimeric peptides are capable of eliciting Th responseswith resultant antibody responses targeted to the B cell site in mostmembers of a genetically diverse population (U.S. Pat. No. 5,759,551).

A review of the known promiscuous Th epitopes shows that they range insize from approximately 15 to 50 amino acid residues (U.S. Pat. No.5,759,551) and often share common structural features with specificlandmark sequences. For example, a common feature is the presence ofamphipathic helices. These are alpha-helical structures with hydrophobicamino acid residues dominating one face of the helix and charged andpolar resides dominating the surrounding faces (Cease et al., 1987).Known promiscuous Th epitopes also frequently contain additional primaryamino acid patterns such as a charged residue, -Gly-, followed by two tothree hydrophobic residues, followed in turn by a charged or polarresidue (Rothbard and Taylor, EMBO J, 1988; 7:93-101). Th epitopes withthis pattern are called Rothbard sequences. It has also been found thatpromiscuous Th epitopes often obey the 1, 4, 5, 8 rule, where apositively charged residue is followed by hydrophobic residues at thefourth, fifth and eighth positions, consistent with an amphipathic helixhaving positions 1, 4, 5 and 8 located on the same face. This pattern ofhydrophobic and charged and polar amino acids may be repeated within asingle Th epitope (Partidos et al., J Gen Virol, 1991; 72:1293-99).Most, if not all, of the known promiscuous T cell epitopes contain atleast one of the periodicities described above.

Promiscuous Th epitopes derived from pathogens include the hepatitis Bsurface and core antigen helper T cell epitopes (HBsAg Th and HBc Th),the pertussis toxin helper T cell epitopes (PT Th), the tetanus toxinhelper T cell epitopes (TT Th), the measles virus F protein helper Tcell epitopes (MVF Th), the Chlamydia trachomatis major outer membraneprotein helper T cell epitopes (CT Th), the diphtheria toxin helper Tcell epitopes (DT Th), the Plasmodium falciparum circumsporozoite helperT cell epitopes (PF Th), the Schistosoma mansoni triose phosphateisomerase helper T cell epitopes (SM Th), and the Escherichia coli TraThelper T cell epitopes (TraT Th). The sequences of thesepathogen-derived Th epitopes can be found in U.S. Pat. No. 5,759,551 asSEQ ID NOS:2-9 and 42-52 therein, incorporated herein by reference; inStagg et al., Immunology, 1993; 79;1-9; and in Ferrari et al., J ClinInvest, 1991; 88: 214-222, also incorporated by reference

The use of such pathogen-derived sites for the immuno-potentiation ofpeptide B cell sites for application to LHRH has been described in U.S.Pat. No. 5,759,551, for HIV in Greenstein et al. (1992), for malaria inEP 0 427,347, for rotavirus in Borras-Cuesta et al. (1987), and formeasles in Partidos et al. (1991).

Useful Th epitopes may also include combinatorial Th epitopes. In Wanget al. (WO 95/11998), a particular class of combinatorial Th epitopes, a“Structured Synthetic Antigen Library” (SSAL) was described. Th SSALepitopes comprise a multitude of Th epitopes with amino acid sequencesorganized around a structural framework of invariant residues withsubstitutions at specific positions. The sequences of the SSAL aredetermined by retaining relatively invariant residues while varyingother residues to provide recognition of the diverse MHC restrictionelements. This may be accomplished by aligning the primary amino acidsequence of a promiscuous Th, selecting and retaining as the skeletalframework the residues responsible for the unique structure of the Thpeptide, and varying the remaining residues in accordance with known MHCrestriction elements. Lists of the invariant and variable positions withthe preferred amino acids of MHC restriction elements are available toobtain MHC-binding motifs. These may be consulted in designing SSAL Thepitopes (Meister et al., Vaccine, 1995; 13:581-591).

The members of the SSAL may be produced simultaneously in a singlesolid-phase peptide synthesis in tandem with the targeted B cell epitopeand other sequences. The Th epitope library sequences are designed tomaintain the structural motifs of a promiscuous Th epitope and at thesame time, accommodate reactivity to a wider range of haplotypes. Forexample, the degenerate Th epitope “SSAL1 TH1” (WO 95/11998), wasmodeled after a promiscuous epitope taken from the F protein of themeasles virus (Partidos et al., 1991). SSAL1 TH1 was designed to be usedin tandem with a target antigen, LHRH. Like the measles epitope fromwhich it was derived, SSAL1 TH1 was designed to follow the Rothbardsequence and the 1, 4, 5, 8 rules and is a mixture of four peptides:

  1               5                  10                  15Asp-Leu-Ser-Asp-Leu-Lys-Gly-Leu-Leu-Leu-His-Lys-Leu-Asp-Gly-Leu (SEQ IDNO:2) Glu Ile     Glu Ile Arg     Ile Ile Ile     Arg Ile Glu     Ile(SEQ ID NO:3)     Val         Val         Val ValVal         Val         Val (SEQ ID NO:4)    Phe         Phe         Phe Phe Phe         Phe         Phe (SEQ IDNO:5)

A charged residue Glu or Asp is added at position 1 to increase thecharge surrounding the hydrophobic face of the Th. The hydrophobic faceof the amphipathic helix is then maintained by hydrophobic residues at2, 5, 8, 9, 10, 13 and 16. Positions at 2, 5, 8, 9, 10, and 13 arevaried to provide a facade with the capability of binding to a widerange of MHC restriction elements. The net effect of the SSAL feature isto enlarge the range of immune responsiveness of the artificial Th (WO95/11998).

Other attempts have been made to design “idealized” artificial Thepitopes” incorporating all of the properties and features of knownpromiscuous Th epitopes. Several peptide immunogens comprising theseartificial promiscuous Th epitopes, including those in the form of SSAL,have also been constructed. The artificial Th sites have been combinedwith peptide sequences taken from self-antigens and foreign antigens toprovide enhanced antibody responses to site—specific targets (WO95/11998; Alexander et al., Immunity, 1994, 1:751; Del Guerio et al.,Vaccine, 1997, 15:441) that have been described as highly effective.Such peptide immunogens are preferred for providing effective and safeantibody responses, and for their immunopotency, arising from a broadlyreactive responsiveness imparted by the idealized promiscuous Th sitesdescribed.

SUMMARY OF THE INVENTION

The present invention provides an immunogenic peptide compositioncomprising a promiscuous artificial T helper cell epitope linked to asynthetic peptide B cell epitope or “target antigenic site”. Theimmunogenic peptide comprise an artificial T helper cell (Th) epitopesand a target antigenic site containing B cell epitopes and, optionally,a general immune stimulator sequence. The presence of an artificial Thepitope in the immunogenic peptide impart thereto a capability to inducea strong T helper cell-mediated immune response with the production of ahigh level of antibodies directed against the “target antigenic site.”The present invention further provides for the advantageous replacementof carrier proteins and pathogen-derived T helper cell sites inestablished peptide immunogens with artificial T helper cell epitopesdesigned specifically to improve their immunogenicity. The novel shortpeptide immunogens with the artificial Th epitopes of the presentinvention elicit a high level of antibodies targeted to luteinizinghormone-releasing hormone (LHRH) useful for contraception, the controlof hormone-dependent tumors, the prevention of boar taint, andimmunocastration.

The artificial Th epitopes were developed empirically, mindful of theknown rules for predicting promiscuous T cell epitopes. In the absenceof a unifying theory explaining the mechanism of Th epitopes, these“predicative” rules serve merely as guidelines for designing effectiveartificial Th epitopes. The artificial Th epitopes of the presentinvention have been found to be useful when linked to target antigenicsites and optionally with a immunostimulatory sequence. The immunogenicpeptides of the present invention may be represented by the formulae:

(A)_(n)-(Target antigenic site)-(B)_(o)-(Th)_(m)-X

or

(A)_(n)-(B)_(o)-(Th)_(m)-(B)_(o)-(Target antigenic site)-X

or

(A)_(n)-(Th)_(m)-(B)_(o)-(Target antigenic site)-X

or

(Target antigenic site)-(B)_(o)-(Th)_(m)-(A)_(n)-X

or

(Th)_(m)-(B)_(o)-(Target antigenic site)-(A)_(n)-X

wherein:

A is an amino acid or a general immunostimulatory sequence, e.g., theinvasin domain (Inv) (SEQ ID NO:78) where n is more than one, theindividual A's may be the same or different;

B is selected from the group consisting of amino acids, —NHCH (X)CH₂SCH₂CO—, —NHCH (X) CH₂SCH₂CO (□N) Lys-, —NHCH (X) CH₂S-succinimidyl(□N) Lys-, and —NHCH (X) CH₂S-(succinimidyl)-;

Th is an artificial helper T cell epitope selected from the groupconsisting of SEQ ID NOS:6-22, 31-35 and 105 and an analog or segmentthereof;

“Target antigenic site” is a desired B cell epitope, a peptide hapten,or an immunologically reactive analog thereof;

X is an aminoacid α-COOH, —CONH₂;

n is from 1 to about 10;

m is from 1 to about 4; and

o is from 0 to about 10.

An example of a peptide hapten as a target antigenic site is LHRH (SEQID NO: 77).

The compositions of the present invention comprise peptides capable ofevoking antibody responses in an immunized host to a desired targetantigenic site. The target antigenic site may be derived from pathogenicorganisms and normally immunosilent self-antigens and tumor-associatedtargets such as LHRH.

Accordingly, the compositions of the present invention are useful inmany diverse medical and veterinary applications. These include vaccinesto provide protective immunity from infectious disease, immunotherapiesfor the treatment of disorders resulting from the malfunction of normalphysiological processes, immunotherapies for the treatment of cancer,and agents to desirably intervene in and modify normal physiologicalprocesses.

Some of the targets antigens which may be covalently linked to the Thepitopes of the present invention include: LHRH for contraception, thecontrol of hormone dependent tumors and immunocastration; somatostatinfor growth promotion in farm animals; IgE for treatment of allergy; theCD4 receptor of T helper cells for treatment and prevention of HIVinfection and immune disorders; foot-and-mouth disease virus capsidprotein as a vaccine for the prevention of foot-and-mouth disease; theCS antigen of Plasmodium for prevention of malaria; CETP for preventionand treatment of arteriosclerosis; and HIV virion epitopes forprevention and treatment of HIV infection.

DETAILED DESCRIPTION OF THE INVENTION

Idealized artificial Th epitopes have been provided. These are modeledon two known natural Th epitopes and SSAL peptide prototypes, disclosedin WO 95/11998. The SSALS incorporate combinatorial MHC molecule bindingmotifs (Meister et al., 1995) intended to elicit broad immune responsesamong the members of a genetically diverse population. The SSAL peptideprototypes were designed based on the Th epitopes of the measles virusand hepatitis B virus antigens, modified by introducing multipleMHC-binding motifs. The design of the other Th epitopes were modeledafter other known Th epitopes by simplifying, adding, and/or modifying,multiple MHC-binding motifs to produce a series of novel artificial Thepitopes. The newly adapted promiscuous artificial Th sites wereincorporated into synthetic peptide immunogens bearing a variety oftarget antigenic sites. The resulting chimeric peptides were able tostimulate effective antibody responses to the target antigenic sites.

The prototype artificial helper T cell (Th) epitope shown in Table 1a as“SSAL1 TH1”, a mixture of four peptides (SEQ ID NOS:2, 3, 4, 5) is anidealized Th epitope modeled from a promiscuous Th epitope of the Fprotein of measles virus (Partidos et al. 1991). The model Th epitope,shown in Table 1a as “MVF Th” (SEQ ID NO:1) corresponds to residues288-302 of the measles virus F protein. MVF Th (SEQ ID NO:1) wasmodified to the SSAL1 Th1 prototype (SEQ ID NOs:2, 3, 4, 5) by adding acharged residue Glu/Asp at position 1 to increase the charge surroundingthe hydrophobic face of the epitope; adding or retaining a chargedresidues or Gly at positions 4, 6, 12 and 14; and adding or retaining acharged residue or Gly at positions 7 and 11 in accordance with the“Rothbard Rule”. The hydrophobic face of the Th epitope compriseresidues at positions 2, 5, 8, 9, 10, 13 and 16. Hydrophobic residuescommonly associated with promiscuous epitopes were substituted at thesepositions to provide the combinatorial Th SSAL epitopes, SSAL1 Th1 (SEQID NOs:2, 3, 4, 5). The hydrophobic residues conforming to the Rothbardsequence rule are shown in bold (Table 1a, SEQ ID NO:1). Positions inthe sequence obeying the 1, 4, 5, 8 rule are underlined. Anothersignificant feature of the prototype SSAL1 Th1 (SEQ ID NOS:2, 3, 4, 5)is that positions 1 and 4 is imperfectly repeated as a palindrome oneither side of position 9, to mimic an MHC-binding motif. This “1, 4, 9”palindromic pattern of SSAL1 Th1 was further modified in SEQ ID NO:3(Table 1a) to more closely reflect the sequence of the original MVFmodel Th (SEQ ID NOs:1). Also, the hydrophobicity of the SSAL1 Th1prototype (SEQ ID NOs:2, 3, 4, 5) was modulated in SEQ ID NOS:6, 7, and8 by the addition of methionine residues at variable positions 1, 12,and 14. Experimental data shows that SEQ ID NOS:6, 7, 8 coupled to atarget antigenic site enhanced the antibody response in the immunizedanimals to the target antigenic site.

SEQ ID NOS:6, 7, 8 was simplified to SEQ ID NOS:6, 9, 10 and 11 (Table1a) to provide further immunogenic SSAL Th epitopes. SEQ ID NOS:6, 7, 8was further simplified to SEQ ID NOS:6, 12-14 (Table 1a) to provide aseries of single-sequence epitopes. SSAL Th SEQ ID NOS:4, 9, 10 and 11and the single sequence Th epitopes SEQ ID NOS:6, 12-14, coupled totarget antigenic sites also provided enhanced immunogenicity

It was found that the immunogenicity of SEQ ID NOS:6, 7, 8 may beimproved by extending the N terminus with a non-polar and a polaruncharged amino acid, e.g., Ile and Ser, and extending the C terminus bya charged and hydrophobic amino acid, e.g., Lys and Phe. This is shownin Table 1a as SEQ ID NO:15, 16 and 17 from which simplified SSAL Thepitopes SEQ ID NOS:15 and 18, 105 and 19 were derived. Peptideimmunogens comprising a target antigenic site and a Th epitope selectedfrom SEQ ID NOS:15-17, 15 and 18, 105 and 19, and 123 and 124 displayedenhanced immunogenicity. Single-sequence peptides such as SEQ ID NOS:15,20-22 were also synthesized and tested for immunogenicity in animals.These were also found to be effective Th epitopes.

The SSAL artificial helper epitope shown in Table 1b as “SSAL2 Th2” (SEQID NOS:26-30) was modeled after a promiscuous epitope from the hepatitisB virus surface antigen SEQ ID NO:23 corresponding to residues 19-33 ofthe hepatitis B surface antigen (HBsAg) (Greenstein et al. 1992). Thepathogen-derived model Th, was modified to SEQ ID NO:24 by adding threeLysines to improve solubility in water; the C-terminal Asp was deletedin SEQ ID NO:25 to facilitate the synthesis of chimeric peptides whereinGly-Gly was introduced as spacers. The SSAL2 Th2 (SEQ ID NOs:26-30) wasfurther modified from SEQ ID NO:24 by varying the positively chargedresidues therein at positions 1, 2, 3 and 5 to vary the chargesurrounding the hydrophobic face of the helical structure. A chargedamino acid at variable position 3 also contributed a required residue togenerate the idealized Th epitope, SSAL2 Th2 (SEQ ID NOS:26-30), whichobeyed the 1, 4, 5, 8 rule (underlined residues). The hydrophobic faceof the amphipathic helix consists of hydrophobic residues at positions4, 6, 7, 10, 11, 13, 15 and 17 of SEQ ID NOS:26-30. The Rothbardsequence residues are shown in bold for prototype SSAL2 Th2 (SEQ IDNOS:26-30).

SEQ ID NOS:31-35 were simplified from the idealized SSAL2 Th2 prototype(SEQ ID NOS:26-30) as described above. For example, variable hydrophobicresidues were replaced with single amino acids, such as Ile or Met (SEQID NOS:31-35). The hydrophobic Phe in position 4 was incorporated as afeature of SEQ ID NO:34 while deleting the three lysines. The deletionof the C-terminal Asp was incorporated as a feature of SEQ ID NOS:32,34, and 35. Further modifications included the substitution of theC-termini by a common MHC-binding motif AxTxIL (Meister et al, 1995).

Each of the novel artificial Th epitopes, SEQ ID NOS:6, 12-19, 105, 123,124 20-22 and 31-35 were coupled to a variety of target antigenic sitesto provide peptide immunogens. The target antigenic sites include thepeptide hormones, LHRH and somatostatin, B cell epitopes fromimmunoglobulin IgE, the T cell CD4 receptor, the VP1 capsid protein offoot-and-mouth disease virus; the CS antigen of Plasmodium falciparum;and cholesteryl ester transport protein (CETP); and B cell epitopes fromHIV. The results show that effective anti-target site antibodiescross-reactive with a diverse group of self-antigens and foreignantigens were produced. Most important, the antibody responses weredirected to the target antigenic sites and not to the novel Th epitopes.The results for the novel peptide immunogens for LHRH are shown inTables 2 and 3. The immunogenicity results also show that the antibodiesproduced were effective against LHRH but not against the Th epitopesthemselves. It is to be emphasized that LHRH is a target antigenic sitedevoid of T cell epitopes (Sad et al., Immunology, 1992; 76: 599-603 andU.S. Pat. No. 5,759,551). Thus, the novel artificial Th epitopes of thepresent invention represent a new class of promiscuous T helperepitopes.

The artificial Th epitopes of the present invention are contiguoussequences of amino acids (natural or non-natural amino acids) thatcomprise a class II MHC molecule binding site. They are sufficient toenhance or stimulate an antibody response to the target antigenic site.Since a Th epitope can consist of continuous or discontinuous amino acidsegments, not every amino acid of the Th epitope is necessarily involvedwith MHC recognition. The Th epitopes of the invention further includeimmunologically functional homologues. Functional Th homologues includeimmune-enhancing homologues, crossreactive homologues and segments ofany of these Th epitopes. Functional Th homologues further includeconservative substitutions, additions, deletions and insertions of fromone to about 10 amino acid residues and provide the Th-stimulatingfunction of the Th epitope.

The promiscuous Th epitopes of the invention are covalently linked tothe N- or C-terminus of the target antigenic site, to produce chimericTh/B cell site peptide immunogens. The term “peptide immunogen” as usedherein refers to molecules which comprise Th epitopes covalently linkedto a target antigenic site, whether through conventional peptide bondsso as to form a single larger peptide, or through other forms ofcovalent linkage, such as a thioester. Accordingly, the Th epitopes(e.g., SEQ ID NOS:6, 12-19, 105, 123, 124, 20-22 and 31-35) arecovalently attached to the target antigenic site (e.g., SEQ ID NO:77)either via chemical coupling or via direct synthesis. The Th epitopesmay be attached directly to the target site or through a spacer, e.g.,Gly-Gly or (□-N)Lys. In addition to physically separating the Th epitopefrom the B cell epitope (e.g., SEQ ID NOS:41-64, 71-76, 84-90, 92-94,96-102, 103, 104, 106, 126-129, and 136-153), the spacer may disrupt anyartifactual secondary structures created by the linking of the Thepitope or its functional homologue with the target antigenic site andthereby eliminate any interference with the Th and/or B cell responses.A flexible hinge spacer that enhances separation of the Th and IgEdomains can also be useful. Flexible hinge sequences are often prolinerich. One particularly useful flexible hinge is provided by the sequencePro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO:79) modeled from the flexible hingeregion found in immunoglobulin heavy chains. Xaa therein is any aminoacid, preferably aspartic acid. The conformational separation providedby the spacer (See SEQ ID NOS:80 and 82) permits more efficientinteractions between the presented peptide immunogen and the appropriateTh cells and B cells. Thus the immune responses to the Th epitope isenhanced to provide improved immune reactivity.

The peptide conjugate immunogens of the invention optionally may alsocomprise a general immunostimulatory peptide sequence. For example, adomain of an invasin protein (Inv) from the bacteria Yersinia spp (Brettet al., Eur J Immunol, 1993, 23: 1608-1614). This immune stimulatoryproperty results from the capability of this invasin domain to interactwith the β1 integrin molecules present on T cells, particularlyactivated immune or memory T cells. A preferred embodiment of theinvasin domain (Inv) for linkage to a promiscuous Th epitope has beenpreviously described in U.S. Pat. No. 5,759,551 and is incorporatedherein by reference. The said Inv domain has the sequence:

Thr-Ala-Lys-Ser-Lys-Lys-Phe-Pro-Ser-Tyr-Thr-Ala-Thr-Tyr-Gln-Phe   (SEQID NO:78)

or is an immune stimulatory homologue thereof from the correspondingregion in another Yersinia species invasin protein. Such homologues thusmay contain substitutions, deletions or insertions of amino acidresidues to accommodate strain to strain variation, provided that thehomologues retain immune stimulatory properties. The generalimmunostimulatory sequence may optionally be linked to the Th epitopewith a spacer sequence.

The peptide conjugates of this invention, i.e., peptide immunogens whichcomprise the described artificial Th epitopes, can be represented by theformulas:

(A)_(n)-(Target antigenic site)-(B)_(o)-(Th)_(m)-X

or

(A)_(n)-(B)_(o)-(Th)_(m)-(B)_(o)-(Target antigenic site)-X

or

(A)_(n)-(Th)_(m)-(B)_(o)-(Target antigenic site)-X

or

(Target antigenic site)-(B)_(o)-(Th)_(m)-(A)_(n)-X

or

(Th)_(m)-(B)_(o)-(Target antigenic site)-(A)_(n)-X

wherein:

A is optional and is an amino acid or a general immunostimulatorysequence, where N>1, each A may be the same or different;

B is selected from the group consisting of amino acids, —NHCH (X)CH₂SCH₂CO—, —NHCH (X) CH₂SCH₂CO (□-N) Lys-, —NHCH (X) CH₂S-succinimidyl(□-N) Lys-, and —NHCH (X) CH₂S-(succinimidyl)-;

Th is an artificial helper T cell epitope (SEQ ID NOS:6, 12-19, 105,20-22 and 31-35) or an immune enhancing homologue or segment thereof;

“Target antigenic site” is a desired B cell epitope or peptide hapten,or an analog thereof);

X is an amino acid α-COOH OR —COHN₂;

n is from 1 to about 10;

m is from 1 to about 4; and

o is from 0 to about 10.

The peptide immunogens of the present invention comprises from about 25to about 100 amino acid residues, preferably from about 25 to about 80amino acid residues.

When A is an amino acid, it can be any non-naturally occurring or anynaturally occurring amino acid. Non-naturally occurring amino acidsinclude, but are not limited gto, D-amino acids, β-alanine, ornithine,norleucine, norvaline, hydroxyproline, thyroxine, γ-amino butyric acid,homoserine, citrulline and the like. Naturally-occurring amino acidsinclude alanine, arginine, asparagine, aspartic acid, cysteine, glutamicacid, glutamine, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine and valine. Moreover, when n is greater than one, and two ormore of the A groups are amino acids, then each amino acid may beindependently the same or different.

When A is an invasin domain sequence, it is preferably an immunestimulatory epitope from the invasin protein of an Yersinia speciesdescribed here as SEQ ID NO:77.

In one embodiment where n is 3, each A is in turn an invasin sequence(Inv), glycine and glycine.

B is optional and is a spacer comprising one or more naturally occurringor non-naturally occurring amino acids. In (B)_(o), where O>1, each Bmay be same or different. B may be Gly-Gly or Pro-Pro-Xaa-Pro-Xaa-Pro(SEQ ID NO:79) or □NLys or —NHCH (X) CH₂SCH₂CO—, —NHCH (X) CH₂SCH₂CO(□NLys)-, —NHCH (X) CH₂S-succinimidyl-□NLys-, and —NHCH (X)CH₂S-(succinimidyl)-.

Th is a promiscuous T helper cell epitope selected from the group SEQ IDNOS:6, 12-19, 105, 123, 124, 20-22 and 31-35 and homologues thereof.

The peptide immunogens of this invention, may be made by chemicalmethods well known to the ordinarily skilled artisan. See, for example,Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed.Grant, W. H. Freeman & Co., New York, N.Y., 1992, p.77. The peptides maybe synthesized using the automated Merrifield solid phase peptidesynthesis with either t-Boc or Fmoc to protect the α-NH₂ or side chainamino acids. Equipment for peptide synthesis are available commercially.One example is an Applied Biosystems Peptide Synthesizer Model 430A or431.

After complete assembly of the desired peptide immunogen, the resin istreated according to standard procedures to cleave the peptide from theresin and de-block the functional groups on the amino acid side chains.The free peptide is purified by HPLC and characterized biochemically,for example, by amino acid analysis, by sequencing, or by massspectometry. Methods of peptide purification and characterization arewell known to one of ordinary skill in the art.

Other chemical means to generate peptide immunogens comprising the Thepitopes of the invention include the ligation of haloacetylated andcysteinylated peptides through the formation of a “thioether” linkage.For example, a cysteine can be added to the C terminus of aTh-containing peptide and the thiol group of cysteine may be used toform a covalent bond to an electrophilic group such as an Nchloroacetyl-modified or a maleimide-derivatized □- or □-NH₂ group of alysine residue, which is in turn attached to the N-terminus of a targetantigenic site peptide. In this manner, Th epitope/B cell siteconjugates may be obtained.

The subject immunogen may also be polymerized. Polimerization can beaccomplished for example by reaction between glutaraldehyde and the —NH₂groups of the lysine residues using routine methodology. By anothermethod, the linear Th/B cell site immunogen can be polymerized orco-polymerized by utilization of an additional cysteine added to theN-terminus of the linear constructs. The thiol group of the N-terminalcysteine can be used for the formation of a “thioether” bond withhaloacetyl-modified amino acid or a maleimide-derivatized □- or □-NH₂group of a lysine residue that is attached to the N-terminus of abranched poly-lysyl core molecule (e.g., K₂K, K₄K₂K or K₈K₄K₂K). Thesubject immunogen may also be polymerized as a branched structurethrough synthesis of the desired peptide construct directly onto abranched poly-lysyl core resin (Wang, et al., Science, 1991;254:285-288).

The longer synthetic peptide conjugates may alternatively be synthesizedby well known nucleic acid cloning techniques. Any standard manual onmolecular cloning technology provides detailed protocols to producepeptides comprising the Th epitopes of the invention by expression ofrecombinant DNA and RNA. To construct a gene expressing a Th/targetantigenic site peptide of this invention (e.g., SEQ ID NOS:36-64, 106,71-76 and 80-82), the amino acid sequence is reverse translated into anucleic acid sequence, preferably using optimized codons for theorganism in which the gene will be expressed. Next, a gene encoding thepeptide is made, typically by synthesizing overlapping oligonucleotideswhich encode the peptide and necessary regulatory elements. Thesynthetic gene is assembled and inserted into the desired expressionvector. The synthetic nucleic acid sequences encompassed by thisinvention include those which encode the Th epitopes of the inventionand peptides comprising those Th epitopes, the immunologicallyfunctional homologues thereof, and nucleic acid constructs characterizedby changes in the non-coding sequences that do not alter the immunogenicproperties of the peptide or Th epitope encoded thereby. The syntheticgene is inserted into a suitable cloning vector and recombinants areobtained and characterized. The Th epitopes and peptides comprising theTh epitopes are then expressed under conditions appropriate for theselected expression system and host. The Th epitope or peptide ispurified and charaterized by standard methods.

Peptide immunogens of the invention may be used alone or in combinationto elicit anitbody responses to Luteinizing Hormone Releasing Hormone.Luteinizing Hormone Releasing Hormone (LHRH) or Gonadotropin-releasinghormone (GnRH) is a master hormone for the regulation of sexualreproduction in both males and females. LHRH regulates the release of LHand FSH which in turn control spermatogenesis, ovulation and estrus,sexual development. LHRH ultimately controls the secretion of the malehormones andorgen and testosterone, and the secretion of the femalehormones, estrogen and progesterone which themselves are essential forfertility in males and females, respectively. (Basic and ClinicalEndocrinology, eds. FS Greenspan and JD Baxter, Appleton andLange:Norwalk Conn. 1994).

Active immunization against LHRH has long been known to exert multipleeffects in males including decreased serum and pituitary LH and FSH,reduced serum testosterone, suppression of spermatogenesis andreversible atrophy of the gonads and accessory sex organs. (See, forexample, Fraser et al., J. Endocrinol., 1974; 63:399-405; Giri et al.,Exp. Molec. Pathol., 1991; 54:255-264; Ladd et al., J. Reprod. Immunol.,1989; 15:85-101; and references cited therein). Immunization againstLHRH has been proven useful as a contraceptive in males and haspotential as a treatment for prostate cancer (Thau, Scand J Immunol,1992; 36 Suppl 11:127-130; and U.S. Pat. No. 5,759,551).

Immune intervention on the hypothalo-pituitary gonadal axis by activeimmunization against LHRH can also be used to inhibit sexual hormones infemales. Since LHRH regulates the production of FSH by the anteriorpituitary which in turn regulates the production of estrogen by theovaries, blocking the action of LHRH is a therapy for sexualhormone-dependent diseases in women. For example, the eptopicdevelopment and maintenance of endometrial tissues outside the uterinemusculature is mediated by estrogen. Therefore, blocking the action ofLHRH is useful as a treatment for endometriosis. Furthermore, by analogyto prostate cancer, estrogen-driven tumors of the breast should also beresponsive to LHRH immunotherapy. Indeed, an anti-LHRH inducing vaccinehas been shown to effectively reduce serum levels of LH and FSH inwomen, an illustration of the potential of this method to effectcontraception and treatment of hormone-dependent disorders (Gual et al,.Fertility and Sterility, 1997; 67:404-407).

In addition to providing treatment for a number of important diseasesand reversible infertility in both men and women, LHRH-basedimmunotherapy provides a means for reversible contraception in male andfemale animals (e.g. dogs, cats, horses and rabbits) as well asmitigating undesirable androgen-driven behavior such as heat,territorial marking and aggression.

Lastly, immunological castration (e.g., antibody-based inhibition ofLHRH action) has application in the livestock industry. Meat from maleanimals is not processed into prime cuts because of the presence of anoffensive aroma and taste, known as boar taint. Boar taint isconventionally eliminated by mechanical castration; however, castrationof male food animals is no longer considered humane. Moreover,mechanical castration results in poorer growth performance and lesionsin body part, also referred to as carcass traits, in comparison tonon-castrated males. Whereas, the growth performance and carcass traitsof immunocastrated animals are less affected than those of castratedanimals (Bonneau et al., J Anim Sci, 1994; 72: 14-20 and U.S. Pat. No.5,573,767). Therefore, immunological castration is preferable tomechanical castration.

LHRH (or GnRH) is a self-molecule that must be linked to a Th componentin order to generate anti-LHRH antibodies (Sad et al., Immunology, 1992;76: 599-603). Several such immunogenic forms of LHRH have been tested.For example, LHRH immunogens have been produced by conjugation tocarrier proteins or linked by peptide synthesis to potent Th sitesderived from pathogenic organisms (WO 94/07530, U.S. Pat. No. 5,759,551,Sad et al., 1992). Improved LHRH peptide immunogens comprising LHRH andartificial Th epitopes are exemplified in Examples 1-3.

This invention also provides for compositions comprisingpharmaceutically acceptable delivery systems for the administration ofthe peptide immunogens. The compositions comprise an immunologicallyeffective amount of one or more of the peptide immunogens of thisinvention. When so formulated, the compositions of the present inventioncomprising LHRH or a homologue thereof as target antigenic site, areused for treatment of prostate cancer, prevention of boar taint,immunocastration of animals, the treatment of endometriosis, breastcancer and other gynecological cancers affected by the gonadal steroidhormones, and for contraception in males and females. The utility forpeptides of the invention having target antigenic sites other than LHRHwill vary in accordance with the specificity of the target antigenicsite.

The peptide immunogens of the invention can be formulated as immunogeniccompositions using adjuvants, emulsifiers, pharmaceutically-acceptablecarriers or other ingredients routinely provided in vaccinecompositions. Adjuvants or emulsifiers that can be used in thisinvention include alum, incomplete Freund's adjuvant (IFA), liposyn,saponin, squalene, L121, emulsigen, monophosphoryl lipid A (MPL),dimethyldioctadecylammonium bromide (DDA), QS21, and ISA 720, ISA 51,ISA 35 or ISA 206 as well as the other efficacious adjuvants andemulsifiers. Such formulations are readily determined by one of ordinaryskill in the art and also include formulations for immediate releaseand/or for sustained release. The present vaccines can be administeredby any convenient route including subcutaneous, oral, intramuscular,intraperitoneal, or other parenteral or enteral route. Similarly theimmunogens can be administered in a single does or multiple doses.Immunization schedules are readily determined by the ordinarily skilledartisan.

The composition of the instant invention contains an effective amount ofone or more of the peptide immunogens of the present invention and apharmaceutically acceptable carrier. Such a composition in a suitabledosage unit form generally contains about 0.5 μg to about 1 mg of thepeptide immunogen per kg body weight. When delivered in multiple doses,it may be conveniently divided into an appropriate amount per dose. Forexample, the dose, e.g. 0.2-2.5 mg; preferably 1 mg, may be administeredby injection, preferably intramuscularly. This may be followed by repeat(booster) doses. Dosage will depend on the age, weight and generalhealth of the subject as is well known in the vaccine and therapeuticarts.

Vaccines comprising mixtures of the subject peptide immunogens,particularly mixtures comprising Th sites derived from both MVF Th,i.e., SEQ ID NOS:6, 12-19, 105, 123, 124, 20-22, and HBsAg Th, i.e., SEQID NOS:31-35, may provide enhanced immunoefficacy in a broaderpopulation and thus provided an improved immune response to LHRH orother target antigenic site.

The immune response to Th/LHRH peptide conjugates or other Th/targetantigenic site conjugates can be improved by delivery through entrapmentin or on biodegradable microparticles of the type described by O'Haganet al. (Vaccine, 1991; 9:768). The immunogens can be encapsulated withor without an adjuvant, and such microparticles can carry an immunestimulatory adjuvant. The microparticles can also be coadministered withthe peptides immunogens to potentiate immune responses

As a specific example, the invention provides a method for inducinganti-LHRH antibody by administering pharmaceutical compositionscomprising Th/LHRH peptide immunogens to a mammal for a time and underconditions to produce an infertile state in the mammal. As used hereinan infertile state is that state which prevents conception. Infertilitycan be measured by methods known in the art, e.g. evaluation ofspermatogenesis or ovulation, as well as by statistical modeling ofexperimental animal data. Other indicators of infertility in malesincludes reduction of serum testosterone to castration levels andinvolution of the testes. The appropriate dose of the composition isabout 0.5 μg to about 1 mg of each peptide per kg body weight. Thisdosage may conveniently be divided into appropriate amounts per dosewhen delivered in multiple doses.

Similarly, the LHRH embodiments of this invention relate to a method fortreating androgen-dependent carcinoma by administering the subjectpeptide compositions to the mammal for a time and under conditions toprevent further growth of the carcinoma. The appropriate unit dose isabout 0.5 μg to about 1 mg of each peptide per kg body weight. This isconveniently divided into the appropriate amounts per application whenadministered in multiple doses.

Further, the LHRH embodiments relate to a method for improving theorganoleptic qualities and tenderness of the meat from male domesticanimals while maintaining the advantageous growth performance of intactmales. The androgenic steroid hormones of intact males are responsiblefor fast growth but their presence is accompanied by non-androgenicsteroids (e.g., 5αandrostenone) and skatole (a product of the microbialmetabolism of tryptophan) which impart unpleasant taste and aroma to themeat. This condition, known as boar taint in the case of swine, detractsfrom the quality of the meat. However, by the active immunization ofyoung males with compositions comprising LHRH peptides of the invention,on a schedule that effects immunocastration in the weeks just prior toslaughter, many of the growth advantages of non-castrated males may beretained while providing meat with improved flavor and tenderness.

The efficacy of the peptide composition of the present inventioncomprising the target antigenic site, LHRH, can be tested by theprocedure described in the Examples 1-3.

Other target antigenic sites which have also been used in peptideimmunogens of the present invention are described in Examples 4-9.Peptide immunogen compositions are useful for inducing immune responsesin mammals against specific target antigens and provide for preventionor treatment of disease, or intervene to usefully modify normalphysiological conditions.

EXAMPLE 1 Immunization of Rats with Peptide Immunogens Containing LHRH

Peptides listed in Tables 2a and 2b were synthesized and tested asdescribed below.

A. Peptide synthesis. The peptides listed in Tables 2a and 2b weresynthesized individually by the Merrifield solid-phase synthesistechnique on Applied Biosystems automated peptide synthesizers (Models430, 431 and 433A) using Fmoc chemistry. Preparation of peptideconstructs comprising structered synthetic antigen libraries (SSALs),e.g., the artificial Th site designated SEQ ID NO:6-8, was accomplishedby providing a mixture of the desired amino acids selected for a givenposition. After complete assembly of the desired peptide orcombinatorial peptides, the resin was treated according to standardprocedures using trifluoroacetic acid to cleave the peptide from theresin and deblock the protecting groups on the amino acid side chains.

The cleaved, extracted and washed peptides were purified by HPLC andcharacterized by mass spectrometry and reverse phase HPLC.

Peptides were synthesized to have the LHRH target antigenic peptide (SEQID NO:77) in tandem with each of the designed Th epitopes as listed inTables 2a and 2b. The Th epitopes were those shown in Tables 1a and 1b(SEQ ID NOS:6, 12-19, 105, 20-22 and 31-35). For purposes of comparison,prior art peptide immunogens comprising model Th sites (SEQ ID NOS:36and 65), and prototype Th sites (SEQ ID NOS:37-40 and 66-70) and apeptide/carrier protein conjugate, KLH-LHRH (Table 2b) were alsosynthesized and tested. The Th/LHRH and Inv/Th/LHRH peptide constructswere synthesized with gly-gly as a spacer between the target antigenicsite and the Th epitope, and with or without gly-gly as a spacer betweenthe Th epitope and the Inv immunostimulatory sequence. In addition, SEQID NOS:80-82 were synthesized with SEQ ID NO: 79 as a spacer between theTh site and the target antigenic site. The results for peptideimmunogens SEQ ID NOS:80-82 are not yet available.

B. Protocols for immunization. The LHRH peptide immunogens shown inTables 2a and 2b were evaluated on groups of 5 to 10 rats as specifiedby the experimental immunization protocol outlined below and beserological assays for determination of immunogenicity on serum samples:

Animals: Sprague-Dawley rats, male

Group Size: 5-10 rats/group

Immunogen: individual peptide immunogen

Dose: amount in μg as specified, in 0.5 mL

Adjuvants:

(1) Freund's Incomplete Adjuvant (IFA); or

(2) Alum (Aluminum hydroxide); One adjuvant per immunogen per group

Dose Schedule: 0, 3, and 6 weeks or 0, 3 weeks as specified

Route: intramuscular

Blood was collected and processed into serum, and stored prior to ELISAand radioimunoassay (RIA) for determination of serum testosteronevalues.

C. Method for determination of immunogenicity. Antibody activities weredetermined by ELISA (enzyme-linked immunosorbent assays) using 96-wellflat bottom microtiter plates which were coated with the LHRH peptide(SEQ ID NO:77) as immunosorbent. Aliquots (100 μ/mL) of the peptideimmunogen solution at a concentration of 5 μg/mL were incubated for 1hour at 37° C. The plates were blocked by another incubation at 37° C.for 1 hour with a 3% gelatin/PBS solution. The blocked plates were thendried and used for the assay. Aliquots (100 μL) of the test immune sera,starting with a 1:100 dilution in a sample dilution buffer and ten-foldserial dilutions thereafter, were added to the peptide coated plates.The plates were incubated for 1 hour at 37° C.

The plates were washed six times with 0.05% TWEEN® in PBS. 100 μL ofhorseradish peroxidase labeled goat—anti—rat IgG antibody was added atappropriate dilutions in conjugate dilution buffer (Phosphate buffercontaining 0.5M NaCl, and normal goat serum). The plates were incubatedfor 1 hour at 37° C. before being washed as above. Aliquots (100 μL) ofo-phenylenediamine substrate solution were then added. The color wasallowed to develop for 5-15 minutes before the enzymatic color reactionwas stopped by the addition of 50 μL 2N H₂SO₄. The A_(492nm) of thecontents of each well was read in a plate reader. ELISA titers werecalculated based on linear regression analysis of the absorbances, withcutoff A_(492nm) set at 0.5. This cutoff value was rigorous as thevalues for diluted normal control samples run with each assay were lessthan 0.15.

D. Determination of immunogen efficacy. Immunogens were evaluated forefficacy by RIA for serum testosterone values. Serum testosterone levelswere measured using an RIA kit from Diagnostic Products (Los Angeles,Calif.) according to manufacturer's instructions. The lower detectionlimit for testosterone ranged from 0.01 to 0.03 nmol/L. Each sample wasanalyzed in duplicate. Serum samples were scored as being at castrationlevel when the testosterone level was below limits of detection and as“near castration” at <0.1 nmol/L. Results were verified by comparison totestosterone levels in serum from mechanically castrated rats.

E. Results. Results from serum samples collected at weeks 10 or 12 arepresented in Tables 2a and 2b. (The peptides of the Tables are orderedby derivation of their Th epitopes, as was done in Tables 1a and 1b.)ELISA data (not shown) demonstrated that immunization by all the listedimmunogens resulted in antibody responses in all animals. The efficacyof the anti-peptide antibody responses, consequential to thecross-reactivity to natural LHRH, was established by determining serumtestosterone levels. Those results are summarized in the right columnsof Tables 2a and 2b as numbers of animals having castration levels serumtestosterone per total animals in the group.

The results shown that the peptides of the invention, whether with astrong adjuvant IFA and administered 3 times at high dose, or with aweek adjuvant Alum and administered twice at low dose were effective inproducing immunocastration. The immunogenicity of the Th sites SEQ IDNOS:6, 9 and 15 were improved by the addition of the Inv domainsequence. See comparisons between SEQ ID NOS:41, 44 and 45, 46 and SEQID NOS:53 and 60. Although, the addition of the Inv domain sequence didnot always result in improvement in immunogenicity, e.g., compare SEQ IDNOS:51 and 52, SEQ ID NOS:61 and 62, and, SEQ ID NOS:74 and 75. Twopeptides of the invention (SEQ ID NOS:50 and 76) were tested only at lowdose with the week adjuvant and failed to cause immunocastration, butthe results with other peptides, e.g., SEQ ID NO:73, indicate that theywould have been effective at a higher dose with a strong adjuvant. Manyof the LHRH peptide immunogens of the present invention weresignificantly more effective at inducing immunocastration than theKLH/LHRH peptide carrier protein conjugate or the peptide immunogenshaving HBsAg Th (SEQ ID NOS:65 or 66-70). See Table 2b.

Also, the peptide immunogens of the present invention were more easilysynthesized than the peptide/carrier conjugate protein or the peptideimmunogens having the more complex prototype Th epitopes of the priorart (SEQ ID NOS: 2-5 or 26-30). Yet, equivalent or improvedimmunogenicity with fewer and lower doses were obtained with the peptideimmunogens comprising the artificial Th epitopes of the presentinvention.

A serological analysis of the antibody responses of rats that hadreceived the LHRH peptides of the invention demonstrated that theanitbody repsonses to the peptides was specifically directed to thetarget antigenic site and not to the novel artificial Th sites. This isa distinct advantage of these peptide immunogens over conventionalpeptide/carrier protein conjugates. Serum samples from rats that hadbeen immunized with the peptides immunogens shown in Table 3, with 25 μgdoses on Alum at 0 and 3 weeks, were compared for reactivities to theLHRH target site and to the Th epitope by ELISAs using the LHRH peptide(SEQ ID NO:77) and the appropriate Th epitope (SEQ ID NO:15 and 18, 31,or 34) as solid-phase substrates in peptide-based ELISAs. Result forthese ELISAs are presented in Table 3 which show that despite high titerresponsiveness to the LHRH moiety of the Th/LHRH peptide conjugates,reactivities for the artificial Th sites were at background levels.

EXAMPLE 2 LHRH Peptide Mixture for Induction of Broader Immunocastrationin Rats

Establishing the relative efficacies of the various artificial Thepitope/LHRH constructs as shown above in Example 1 permitted selectionof the most effective ones for assembly into a peptide mixture ofenhanced immunogenicity. A mixture of Th/LHRH peptide immunogens is moreefficacious than any individual peptide within the mixture (U.S. Pat.No. 5,795,551). Moreover, a mixture of individual constructs carryingpromiscuous Th epitopes derived from MVF Th (SEQ ID NO:1) and HBsAg Th(SEQ ID NOS:23-25) provide broader response in a genetically diversepopulation than would a peptide composition having Th epitopes derivedfrom only one promiscuous Th epitope. Therefore, a peptide compositioncomprising a mixture of peptides of the invention derived from MVF Thand HBSAg Th was assembled and the efficacy of the mixture was testedand compared to compositions comprising the individual peptides of themixture.

Groups of 6 or 8 male rats were immunized with 25 μg doses (total dose)of the peptide compositions indicated in Table 4. The peptides in themixture were combined in equimolar proportions. The peptides wereformulated with 0.4% Alum and administered intramuscularly on weeks 0and 3. Serum testosterone levels were followed for 22 weeks and theresults were scored as number of animals with castration level oftestosterone per total number of animals in the group. These results arepresented in Table 4. The demonstrated that the low doses of peptidecompositions, given with a relatively ineffective adjuvant, achievedcastration levels of testosterone by week 5, and that this response wasmaintained through week 22. Moreover, the peptide mixture performedsignificantly better than one of the peptide compositions comprising anindividual peptide. It can be assumed that the mixture would have shownimproved immunogenicity over the other individual peptide compositionhad the numbers of experimental animals been larger and morerepresentative of a true population.

EXAMPLE 3 LHRH Peptide Mixture and Formulations for the Immunocastrationof Swine

A group test animals have been shown to be more broadly responsive to amixture of peptide immunogens with different Th epitopes than to acomposition containing a single peptide immunogen. However, for theprevention of boar taint in swine, it is necessary that the immunopotentLHRH peptide immunogens be sufficiently potent to elicit the desiredresponse in most animals while being acceptable for use in food animals.It is important that there is no immediate effect adverse to the growthrate and that no residue of the peptide immunogen or the adjuvant isleft in the meat or cause lesions in the marketable parts of thecarcass.

In order to evaluate the useful immunogenicity of a mixture of inventiveLHRH peptides, the mixture was administered to swine in threeformulations wither in 0.4% Alum, IFA, or ISA 206/DDA. ISA 206/DDA is anoil/water emulsion in which Dimethydioctadecylammonium bromide (DDA) isdispersed into MONTANIDE® ISA 206 at 30 mg/mL (MONTANIDE® ISA 206 is anoily metabolizable solution supplied by SEPPIC Inc. of Fairfield, N.J.).The oil suspension is then emulsified at a 1:1 volume ratio into anaqueous peptide solution which has been adjusted for peptideconcentration so as to provide the desired dose of peptide in 05 mL ofthe final preparation.

The immunization protocol was as follows:

Animals: Yorkshire Hampshire Cross Swine, males, 3-4 weeks of agenon-castrated

Group Size: 2-3 animals/group

Immunogen: Equimolar mixture of SEQ ID NOS:57-58, 71 and 75

Dose: 400 μg of peptide(s) in 0.5 mL

Adjuvants:

(1) 0.4% Alum,

(2) IFA,

(3) ISA 206/DDA

Schedule: 0, 4, and 13 weeks or 0, 4 weeks

Route: Intramuscular

The efficacy of the peptide immunogen formulations was monitored byassaying the swine serum samples collected throughout the course of thestudy. The assays included an RIA for the determination of the presenceof antibodies cross-reactive to native LHRH in solution as describedbelow, and an RIA for testosterone as described in Example 1. Further,the average testes cross sectional area was determined by palpitationwith a caliper.

Antisera for the anti-LHRH RIA were diluted 1:100 in 1% bovine serumalbumin (BSA), pH 7.4. An equal volume of diluted sera was added to 100μL of [¹²⁵I]-LHRH (New England Nuclear Company, Boston, Mass.) dilutedin 1% BSA to contain approximately 15,000 cpm for 5.25 pg LHRH. Thesolution was incubated overnight at room temperature and antibody-boundLHRH was precipitated with 400 μL of 25% polyethylene glycol (MW 8,000)in 0.01M phosphate-buffered saline (PBS), pH 7.6, and 200 μL of 5 mg/mLbovine gamma globulin in PBS. Anti-LHRH antibody concentrations areexpressed as nmol iodinated LHRH bound per liter of serum (Ladd et al.,1988, Am J Reprod Immunol, 17:121-127).

The alum preparation was least effective producing a lower level ofantibody responses. One animal of this group did not achieve thecastration level of testosterone until week 11 and both animals in thisgroup did not manifest complete involution of the testes. The animals ofthe alum group did not receive immunizations at week 13, and the effectsof the treatment were reversed.

The animals of the IFA group displayed higher levels of antibodyresponses, with two of the three reaching and holding a castration levelof testosterone by week 6. However, upon administration of a boosterdose at week 13, the lowest responding swine of the three failed torespond and reverted to a normal levle of testosterone and tonon-involuted tests. The two responsive animals of this group achievedcomplete involution of the testes by week 23.

Both swine of the ISA 206/DDA group provided high and relatively uniformlevels of antibody responses. Immunocastration levels of testosterone inthis group were achieved by week 9 and stably maintained through week12. Both animals were responsive to the boost at week 13 and maintainedcastration levels of testosterone. The testes of both animals wereundetectable by week 23.

From the results obtained the ISA 206/DDA formulation is, thus, mostpreferred for prevention of boar taint. High and uniform effects on thetwo animals are achieved with the ISA 206/DDA formulation. Moreover, theformulation is more acceptable in swine in comparison to the IFAformulation which caused lesions, apparently because the IFA formulationis not readily metabolized.

EXAMPLE 4 Somatostatin Immunogens for Growth Promotion in Farm Animals

Immunogens of the invention may be used singly or in combination toelicit antibodies to somatostatin. Somatostatin is a major inhibitor oftotal somatic growth. It is a cyclic peptide hormone of fourteen aminoacids (SEQ ID NO:80, Table 5) and its structure is conserved acrossspecies. Somatostatin inhibits the release of many gastro-intestinalhormoes as well as inhibits the release of growth hormone, insulin, andthyroid hormones, thereby affecting both the ability of the animal toabsorb nutrients and its subsequent ability to direct these nutrientsinto tissue growth. The neutralization of somatostatin by immunizationhas been shown to stimulate growth in sheep, goats, chickens, and pigs(Spencer, Dom Anin Endocr, 1986; 3:55; Spencer et al., Reprod NutrDevelop, 1987; 27(2B):581; Laarveld et al., Can J Anim Sci, 1986,66:77), and cattle (Lawrence et al., J Anim Sci, 1986, 63(Suppl):215).

In addition to stimulating growth rate and leading to a 20% reduction inrearing time (Spencer, 1986; Spencer et al., 1987) active immunizationagainst somatostatin also has a beneficial effect the efficiency of foodconversion, i.e., in addition to the saving on feed by virtue of morerapid growth, the animals actually utilize their food more efficientltyduring the growing period, at least partly as a result of changes in gutmotility (Fadlalla et al., J Anim Sci, 1985, 61:234). The treatment doesnot have any marked effect on carcass composition (Spencer et al., 1987)but there were indications that, when killed at equal weights, treatedanimals may be less fatty and leaner. Taken all of the experimental datatogether, effective active immunization to somatostatin (as evidenced bythe presence of anti-somatostatin antibodies) is a powerful, safe, andeffective tool to enhance growth (Spencer, 1986).

However, somatostatin is a short peptide and a self-antigen and isnon-immunogenic by itself (see Table 5). Nevertheless, severalimmunogenic forms of somatostatin have been designed and tested asreported in the literature. For example, somatostatin has beenconjugated with protein carriers to enhance immunopotency. However,protein carriers are too expensive for economical use in farm animals.Further, effective immunization with somatostatin is highly dependent onhow the carrier is conjugated to somatostatin. In most cases,glutaraldehyde is employed as the carrier for coupling with the lysineresidues present on somatostatin and glutaraldehyde The two lysines onsomatostatin available for coupling reside within a 12-mer functionalloop. The conjugation of these lysines may result in significant loss ofthe native somatostatin structure. As a result, cross-reactivity tonatural somatostatin with the anitbodies is reduced. Moreover, withprotein carriers, the majority of immune responses are directed to thecarrier rather than to somatostatin (the mass of the carrier molecule(s)is much greater than that of somatostatin). Immunization with a smallpeptide carrier conjugates has frequently led to carrier-induced immunesuppression (Schultz et al., J Immunol, 1985, 135:2319). Accordingly,there is a need for a different way to enchance the immunogenicity thatis more suitable for farm animal use. The vaccine should be inexpensiveand capable of stimulating an early and strong immune response tosomatostatin and avoid carrier-induced suppression.

The somatostatin/Th epitope peptide immunogens shown in Table 5 weresynthesized and administered to rats. The effect of the immunization wasdetermined by peptide ELISA as described in Example 1. CyclizedSomatostatin was used in one of the peptide immunogen (SEQ ID NO:80)tested and in the assay as the solid-phase substrate in ELISA. Tocompletely cyclize somatostatin, the cleaved peptide was dissolved in15% DMSO in water for 48 hrs to facilitate intra-disulfide bondformation.

From the results shown in Table 5, it is clear that somatostatin aloneis devoid of immunogenicity whereas the peptide immunogens of thepresent invention elicited high titers of somatostatin-specificantibodies in the immunized hosts. The anti-somatostatin responsesgenerated for SEQ ID NO:81-83, SEQ ID NO:84-86 and SEQ ID NO:87, with Thepitopes (SEQ ID NOS:6,7,8 and 31) show the effectiveness of the Thepitopes. However, a close comparison of the antibodies titers forimmunogenicity show that it is preferable to place the Th epitope on theC-terminus. The results for SEQ ID NOS:84-86, with the Th epitope as SEQID NOS:6,7,8 shows an earlier higher level of antibodies was elicited.The results of Table 5 illustrate the strong antibody response toimmunization with artificial Th/somatostatin compositions, therebyestablishing the utility of these peptides of this invention for growthpromotion in farm animals.

EXAMPLE 5 Peptide Composition for Prevention of HIV Infection

Peptide immunogens comprising idealized artificial Th sites of thepresent invention and a target antigenic site cross-reactive to a hostcell receptor/co-receptor complex for HIV may be used to elicitantibodies to that host cell complex in the immunized host. Said complexwhich is located on the surface of host lymphocytes expressing CD4comprise CD4 associated with a chemokine receptor domain. This complexis the primary receptor for entry of HIV into T cells. Antibodiesdirected to this CD4 complex block the iteractions between HIV and itsreceptor, and the interactions between CD4-Class II and CD4-expressing Tcells and other activated T cells. Thus, antobodies directed to thiscomplex have broad neutralizing activities against primary isolates ofHIV-1, HIV-2, and SIV and intervene in the immunosuppression ofCD4+cell-mediated immune responses (WO 97/46697).

The peptide immunogens relevant to the CD4 complex antigenic site may beformulated singly or in combination for the generation, by activeimmunization in mammals including humans, high titers of serumantibodies to the CD4 complex. These antibodies are useful for theprevention and treatment of immunodeficiency virus infection as well asfor treatment of undesirable immune responses such as transplantrejection, rheumatoid arthritis, systemic lupus erythematosis, andpsoriasis.

An artificial Th epitope/target antigenic site peptide immunogen wasdesigned with a sequence modified from the CDR2-like domain of CD4 asthe target antigenic site. The modified site comprises a peptidesequence taken from the CDR2-like domain of human CD4 (amino acids 39-66according to the numbering system of Maddon et al., Cell, 1985; 42:93;and, Littman et al., Cell, 1988; 55:541) modified as follows: (1) theinsertion of cysteine residue to the N-terminus side of position 39 ofthe naturally occurring CD4 sequences, (2) the insertion of a cysteineresidue at the C-terminus side of position 66 thereof, and (3) theformation of a disulfide bond between the inserted cysteines to producea cyclic structure. The optimized and modified (i.e., cyclized) targetsite for CD4-CDR2 of the following sequence is provided:

1.Cys-Asn-Gln-Gly-Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser-Lys-Leu-Asn-Asp-Arg-Ala-Asp-Ser-Arg-Arg-Ser-Leu-Trp-Asp-Gln-Gly-Asn-Cys

2. (SEQ ID NO:88)

To complete the cyclization, the modified peptide was dissolved in 15%DMSO in water for 48 hrs to facilitate intra-disulfide bond formationbetween the cysteines. SEQ ID NO:84 was incorporated into the peptideimmunogen:

(SEQ ID NO:6,7,8)-GG-(SEQ ID NO:88) (SEQ ID NO:89-91)

Immunogenicity in guinea pigs of SEQ ID NO:85 formulated in ISA 206/DDA,100 μg/dose, given at weeks 0, 3, 6, was evaluated. Immunogenicity wasdetermined by peptide ELISA as described in Example 1 The cyclizedtarget antigenic site peptide used as the solid-phase substrate in theELSIA. The labeled conjugate was specific for guinea pig IgG. Six of sixguinea pigs were successfully seroconverted by the ELISA reactivelyobtained. It is significant that SEQ ID NO:85 was found to be highlyimmunogenic and is functional in a large animal.

An immunogenic composition comprising SEQ ID NO:85 was formulated inIFA, 300 μg/dose, and administered to a swine by intramuscular injectionon weeks 0, 3, and 6. The swine seroconverted and the serum from week 8was tested for neutralization acitivity against a primary isolate ofHIV-1. Neutralization activity was assayed on HIV-1 VL135, a primaryisolate of subtype B, by MT-2 Microplaque Neutralization Assay (Hansonet al., J Clin Microbiol, 1990; 28:2030; WO 97/46697). The swine serumsample provided 50% virus neutralization at a dilution of 1:249, and 90%neutralization at a dilution of 1:97. Therefore, immunization of a largeanimal host with a peptide immunogen composition of the presentinvention produced antibodies which bind to the host cell receptorcomprising CD4 and neutralize HIV.

EXAMPLE 6 Peptide Composition for Treatment of Allergy

Peptide immunogens comprising the idealized artificial Th sites of theinvention and a target antigenic site that cross-reacts with an effectorsite on the third constant domain (CH3) of the epsilon (Ε) heavy chainof IgE are provided. The immunogens may be used to elicitauto-antibodies to the IgE effector site in the immunized host. That IgECH3 effector site is modified from a segment of the CH3 domain of theepsilon (Ε) heavy chain of human IgE (amino acids 413-435 (Dorringtonand Bennich, 1978; 41:3). It is modified from that of the naturallyoccurring IgE sequence as follows: (1) insertion of a cysteine residueto the N-terminus side at position 413. (2) substitution/for the nativecysteine at position 418 of the native IgE sequence by serine, (3)insertion of cysteine at C-terminus side at position 435, and (4)formation of a disulfide bond between the cysteines at the N- and C-termini to produce a cyclic structure. By this process the targetantigen site for human IgE is optimized.

3. Cys-Gly-Glu-Thr-Tyr-Gln-Ser-Arg-Val-Thr-His-Pro-His-

4. Leu-Pro-Arg-Ala-Leu-Met-Arg-Ser-Thr-Thr-Lys-Cys (SEQ ID NO.:92)

Amino acid substitutions from the natural sequence are shown inboldface. The results show that the polyclonal antibodies elicited havespecificity for the CH3 effector site of IgE in the immunized host. Theyprevent the sensitization of mast cells and basophils by IgE, therebypreventing the triggering and activation of mast cells/basophils andleading to the down-regulation of IgE synthesis. Moreover, antibodieselicited by peptides shown in Table 6 comprising this target antigenicsite are cross-reactive with human IgE, and these antibodies are safeand non-anaphylactogenic. Furthermore, the antibodies do not crosslinkto IgE bound to the high affinity cell receptor to induce degranulation.

The peptide conjugates relevant to the IgE-CH3 antigenic site may beformulated singly or in combination and used to immunize mammalsincluding humans to generate high titers of serum antibodies, which areuseful for the prevention and treatment of allergic symptoms.

The peptide immunogens incorporating the modified IgE-CH3 target site(SEQ ID NO:92) are shown in Table 6 as SEQ ID NOS:87-90. These peptideimmunogens were used to immunize groups of 3 guinea pigs using 100μg/dose, formulated in CFA at week 0, IFA at weeks 3 and 6, andadministered intramuscularly. For comparison, a group of two animalswere immunized with a peptide/KLH carrier protein conjugate at 200μg/dose, similarly administered. ELISA results of serum samplescollected at week 8 are shown. In the ELISA a human IgE myeloma protein(American Biosystem, Inc. cat no. A113) was used as the solid-phaseimmunoadsorbant. The procedure used was identical to the peptide-basedELISAs described previously, except IgE myeloma was used as the solidphase immunoadsorbant. Thus, the results demonstrate cross-reactivitywith human IgE. The ELISA results also demonstrate that all of theconstructs were immunogenic with cross-reactivity for human IgE, withthe peptide immunogens of the present invention providing superiorimmunogenicity. The inclusion of the Inv domain sequence (SEQ ID NO:78)augmented the immunogenicity, as shown by the enhanced immunogenicity ofSEQ ID NO:99 over SEQ ID NO:98.

To test the anti-human IgE antibodies in assays for biological activityand safety, guinea pig hyperimmune sera were produced against SEQ IDNO:98 and 99. The procedure is as described above, except that theanimals also received a booster dose of the peptide immunogen in IFA onweek 10. Guinea pig IgG antibodies were purified and the ability of thepurified antibodies to inhibit the sensitization of human basophils byallergen-specific IgE determined as follows.

Guinea pig IgG antibodies were purified by Protein A affinitychromatography (ImmunoPure® Immobilized Recomb® Protein A, Pierce) fromserum collected at weeks 8 and 12. The serum from the animals immunizedwith SEQ ID NOS 98, 99 were pooled. The eluted antibodies were preparedat a standard concentration of 8 mg/mL in 25 mM PIPES buffer, 0.15 MNaCl, pH 7.2. A control antibody preparation was prepared from thepooled serum of guinea pigs immunized with an irrelevant peptideimmunogen. These antibodies were used in assay that measure thereduction in IgE-mediated sensitization of human basophils. Humanbasophils were prepared from the venous blood of volunteers usingcentrifugation through Percoll density gradients (MacGlashan. J AllergyClin Immunol, 1993; 91:605-615). The banded leukocytes were collected,washed, and resuspended in 0.1 mL of PAGCM buffer as describe(MacGlashan, 1993) except that the PAGCM buffer used to suspend thecells was made up with water containing 44% D₂0. The IgE used for theassay was allergen-specific, either human BPO-specific IgE or chimerichuman IgE having grafted variable domains with specificity for HIVglycoprotein gp120. The allergen-specific IgE at 025 μg/mL waspreincubated for 15 minutes at 37° C. with an equal volume of thepurified antibody at 8 mg/mL. The total volume was 0.1 mL. The antibodymixture was added to the and incubated for 20 minutes to allow forsensitization of the basophils by uncomplexed IgE. The sensitized werethen stimulated by addition of the allergen, either BPO₂₁-HSA or a gp120polypeptide as described (MacGlashan, 1993). After an appropriateincubation period (usually 45 minutes), the basophils were separatedfrom the supernatant and the supernatant assayed for histamine contentby an automated fluorimetric technique (Siraganian, Anal Biochem, 1974;57: 383-394). All reactions were performed in duplicate. The percenthistamine release was calculated from the ratio of sample of totalhistamine minus from both the amount of spontaneous histamine release.The histamine release by experimental antibody to histamine release bythe control antibody of irrelevant specificity was compared and theratio obtained. (Histamine release assay on human basophils were kindlyperformed under coded conditions by Dr. Donald W. MacGlashan, the JohnsHopkins University School of Medicine, Johns Hopkins Asthma and AllergyCenter, Baltimore). Specific inhibition of histamine release by thesite-specific anti-IgE was 61% and 71% for the antibodies purified frombleeds taken on weeks 8 and 12, respectively.

These actively induced polyclonal antibodies were then further testedfor safety. They were tested for ability or inability to cross-linkreceptor-bound IgE and induce spontaneous histamine release in theabsence of allergen. This establishes whether or not they arenon-anaphylactogenic anti-IgE antibodies. A preparation of guinea piganti-SEQ ID NO:98 was tested by direct challenge of IgE-sensitizedbasophils, in the absence of allergen, to evaluate its ability tocrosslink receptor-bound IgE and induce degranulation. Histamine releaseby anti-SEQ ID NO:98 was equivalent to the level of spontaneoushistamine release by the donor cells. Based on this result, it wasconcluded that antibody of specificity for the target antigen site ofSEQ ID NO:98, i.e, SEQ ID NO:92, is non-anaphylactogenic.

The 12 week anti-SEQ ID NO:98 preparation was also evaluated forIgE-specificity, to determine the potential of these antibodies forisotype specific immunosuppresion. The cross-reactivity of anti-SEQ IDNO:98 guinea pig antibodies to human IgE and IgG were compared by ELISA.The procedure is described for human IgE. For human IgG ELISA, human IgGwas used as the solid-phase immunosorbent.

The IgE ELISA plates were coated with the human IgE myeloma at 5 μg/mL.For the IgG ELISA, the plates were coated with human purified IgG (Sigmareagent grade human IgG), also at 5 μg/mL. The purified guinea piganti-SEQ ID NO:98 was tested for reactivity in both ELISAs atconcentrations of 0.5 and 0.1 μg/mL. Results were compared to antibodiespurified from control guinea pig serum and to a “no antibody” control.The A₄₉₀ values obtained for anti-SEQ ID NO:98 antibody on IgE were1.126 at 0.5 μg/mL and 0.344 at 0.1 μg/mL. The A₄₉₀ values obtained foranti-SEQ ID NO:98 antibody on IgG were equal to control antibody andbackground values. This shows that there was no cross reactivity of theguinea pig anti-SEQ ID NO:98 to human IgG.

The peptide immunogen composition of the present invention did notelicit antibodies that recognize IgG antibodies, and therefore areisotype specific for IgE. Thus, it can be concluded that activeimmunization with Th/IgE target antigenic site immunogens of theinvention elicit safe non-anaphylactogenic anti-IgE antibodies. Theantibodies were effective in inhibiting IgE-mediated sensitization, anddisplayed an immunosuppressive potential specific to antibodies ofisotype IgE.

EXAMPLE 7 Peptide Composition for Prevention of Foot-and-Mouth Disease

Peptide immunogens comprising idealized artificial Th sites of theinvention and a target antigenic site cross-reactive to the “G-H Loop”on the VP1 capsid protein of Foot-and Mouth Disease Virus (FMDV), may beused to elicit neutralizing antibodies to FMDV.

Foot-and-mouth disease (FMD) is the most economically important diseaseof domestic livestock. Cloven-hoofed species including cattle, pigs,sheep and goats are susceptible. Seven distinct serotypes have beendescribed: A, O, C, Asia, and the South African types SAT-1, 2, and 3,each of which can be subdivided into multiple subtypes. Viruses ofserotypes A, O, and Asia-1 are most common. Serotype A viruses are themost variable, having more than 30 subtypes. There is nocross-protection between serotypes so that animals recovered frominfection with or vaccinated against a virus of one serotype are stillsusceptible to infection with viruses from the remaining six serotypes.Moreover, the degree of antigenic variation within a serotype is suchthat a vaccine made against one subtype may not be protective foranother subtype within the same serotype (Brown, Vaccine, 1992;10:1022-1026).

Serotype-specific peptides corresponding to the 141-160 region (the G-HLoop) of the VP1 capsid proteins of isolates belonging to all seven FMDVserotypes have been shown to elicit protective levels of type-specificneutralizing antibodies in guinea pigs (Francis et al., Immunology,1990; 69:171-176). Clearly this region contains a dominant immunogenicsite which also carries the serotype specificity of the virus. Theinitial observations of immunogenicity for these 141-160 VP1 peptideswas accomplished using synthetic peptides conjugated to carrier proteinKLH (keyhole limpet hemocyanin), a procedure which negates the advantageof manufacturing a well-defined synthetic immunogen. However, thedevelopment of VP1 synthetic immunogens was advanced by DiMarchi andBrook (U.S. Pat. No. 4,732,971) who showed that two VP1 sequences from asubtype O isolate, joined in a 200-213 Pro-Pro-Ser-141-158-Pro-Cys-Gly(SEQ ID NO:100) chimeric construct, elicited antibody levels whichprotected cattle against challenge. Nevertheless, the efficiency of thiseffect was limited by the low immunogenicity of the peptide immunogenand by its narrow serotype specificity. Practical application demandsthat a vaccine formation provide protection from both homotypic andheterotypic exposure, with small amounts of peptide immunogen.

The G-H Loop immunodominant site optimized for immunogenicity andcapacity to induce broadley neutralizing antibodies is incorporated as atarget antigenic site into the peptide immunogens of the presentinvention. The site is homologous to amino acid positions 134-169 on theFMDV VP1 protein of strain A12 (Robertson et al, J Virol, 1985;54:651-660) and extend beyond either end of the G-H Loop. The targetsite was further modified by: (1) substitution of Asp at position 134and Gln at position 157 with cysteines, (2) formation of a disulfidebond between the substituent cysteines to produce a cyclic structure,and (3) construction of an SSAL thereof using the corresponding VP1sequences from a selection of subtypes. The optimized target antigensites for the FMDV VP1 are exemplified by the peptides listed in Table7. The two peptide immunogens, SEQ ID NO:101 and SEQ ID NO:102, are SSALtarget antigenic sites compiled from strains of FMDV serotypes O andAsia, respectively. The target antigenic sites of both serotype O andAsia were coupled to the Th epitodes of the present invention, SEQ IDNO: 31. The Inv domain sequence (SEQ ID NO:78) was also incorporatedinto SEQ ID NO:102.

These two SSAL peptide immunogens were synthesized and used tohyperimmunize groups of three Duncan Hartley guinea pigs (female, 9weeks old, 450 gm, virus free). Each animal was immunized with 100 μgper dose of the indicated synthetic construct emulsified in CFA at week0 or IFA at weeks 3 and 6. The animals were bled on weeks 0, 5 and 10for testing.

Serum samples were obtained from five and 10 week bleeds and pooled fromeach group. The pooled sera were evaluated for reactivity to the VPIneutralizing epitope by peptide based ELISA using a peptide having thesequences for the neutralizing epitope (VP1 134-169) as the solid phaseimmunoadsorbant. Their ability to neutralize FMDV strains A1 A12_(FP),A_(FL), A23, O-1_(JH), O-1_(P2), and Asia-1 were also determined. Thevirus neutralizing activity of a 1:100 dilution of a serum sample wasdetermined by observing neutralization on a series of increasing inputviral loads, using aliquots (10,000 MPD₅₀) of the virus strains namedabove. The results are shown in Table 7. The testing method performed(Morgan and Moore, Am J Vet Res, 1990; 51:41-45), demonstrated thatimmunization with the peptide antigens of the present invention providedsera which reduced by 2.5 Log₁₀ FMDV microplaques at 1:100 dilution. Theresults also are highly predictive of protective immunity against FMDVinfection.

As shown by Table 7, high anti-peptide titers against the targetantigenic sites (>5 Log₁₀) were elicited by week 5. Broad and effectiveneutralization of all seven tested strains belonging to three differentFMDV serotypes (i.e., A, O, Asia) were observed, despite the widevariations between these strains and serotypes.

This further demonstrates the efficacy of the artificial Th epitopes ofthe present invention to stimulate effective antibody responses againstan epitope from a foreign pathogen.

EXAMPLE 8 Peptide Composition of a Sporozoite Malaria Vaccine

Peptide immunogens comprising the idealized artificial Th sites and acircumsporozoite (CS) target antigen from Plasmodium falciparum, a humanmalaria parasite, are provided. The CS protein is the major surfaceantigen of the sporozoite stage of the parasite. Immunologic studies andsequence data on a large number of CS genes of human, simian, and rodentplasmodia have been demonstrated showing that all CS proteins contain acentral region, consisting of a series of tandem repeats, encompassingmultiple copies of an immunodominant B cell epitope. In P. falciparumthe repeating epitope is represented as

(Asn-Ala-Asn-Pro)_(n) (SEQ ID NO:103)

Antibodies directed against the repeats of the CS protein of the humanmalaria parasites, P. falciparum and P. vivax, inhibit the invasion ofhepatocytes by sporozoites and abolish their infectivity. Therefore,repeats of the CS epitope have been the target antigen of subunitvaccines used in various human malaria vaccine trials (Nussenzweigh, etal., Adv. Imunol., 1989, 45:283; Hoffman et al., Science, 1991,252:520). However, one of the shortcomings of the various syntheticmalaria vaccines currently in clinical trials is their lowimmunogenicity. Thus, a potential malaria vaccine remains to bedeveloped. (Calvo-Calle et al., J Immunol, 1994, 150:1403)

To overcome the immunogenicity problem associated with the repeats ofthe CS protein of P. falciparum, the artificial Th epitopes shown inTable 1 are incorporated into the CS peptide immunogens. For example,peptide construct

(SEQ ID NOS:15,18)-□NLys-(Asn-Ala-Asn-Pro)₄ (SEQ ID NOS:104,105)

is synthesized and used as the key immunogen in a malaria vaccine toelicit potent protective antibodies in small animals, primates (e.g.,baboons) and human against P. falciparum sporozoites.

EXAMPLE 9 Peptide Composition for Prevention and Treatment ofAtherosclerosis and Cardiovascular Disease

Cholesteryl ester transport protein (CETP) mediates the transfer ofcholesteryl esters from HDL to TG-rich lipoproteins such as VLDL andLDL, and also the reciprocal exchange of TG from VLDL and LDL (Tall, JInternal Med, 1995, 237:5-12; Tall, J. Lipid Res, 1993, 34:1255; Hesleret al., J. Biol Chem, 1987, 262:2275; Quig et al., Ann Rev Nutr, 1990,10:169). CETP may play a role in modulating the levels of cholesterylesters and triglyceride associated with various classes of lipoproteins.A high CETP cholesteryl ester transfer activity has been correlated withincreased levels of LDL-associated cholesterol and VLDL-associatedcholesterol, which in turn are correlated with increased risk ofcardiovascular disease (see, e.g., Tato et al., Arterioscler ThrombVascular Biol, 1995, 15:112).

Cetp isolated from human plasma is a hydrophobic glcycoprotein having476 amino acids. A cDNA encoding human CETP has been cloned andsequenced (Drayna et al., Nature 1987, 327:632). A monoclonal antibodyTP2 (formerly designated 5C7) has been produced which completelyinhibits the cholesteryl ester and triglyceride transfer activity ofCETP and, to a lesser extent, the phospholipid transfer activity(Hesler, et al., J Biol Chem, 1988, 263:5020). The epitope of TP2 waslocalized to the carboxyl terminal 26 amino acids, i.e., the amino acidsfrom Arg451 to Ser476 of human CETP (see, Hesler et al., 1988):

Arg Asp Gly Phe Leu Leu Leu Gln Met Asp Phe Gly Phe Pro Glu His Leu LeuVal Asp Phe Leu Gln Ser Leu Ser (SEQ ID NO:106)

TP2 was reported to inhibit both human and rabbit CETP activity in vitroand rabbit CETP in vivo (Yen et al., J Clin Invest, 1989, 83:2018).Further analysis of the region of CETP bound by TP2 revealed that aminoacids between Phe463 and Leu475

Phe Gly Phe Pro Glu His Leu Leu Val Asp Phe Leu Gln Ser Leu Ser

(SEQ ID NO:107)

are necessary for TP2 binding and for neutralizing CETP neutral lipidbinding and transfer activity (see, Wang et al., J Biol Chem, 1992,270:672).

A number of in vivo studies utilizing animal models or humans haveindicated that CETP activity can affect the level of circulatingcholesterol-containing HDL. Increased CETP cholesteryl ester transferactivity can produce a decrease in HDL-C levels relative to LDLC and/orVLDLC levels and low HDL which in turn is correlated with an increasedsusceptibility to arteriosclerosis. Therefore, the discovery ofcompounds and methods to control CETP activity will be advantageous inpreventing or treating cardiovascular disease.

Rittershaus et al., WO 96/34888, proposed the use of a peptidecomposition comprising a “universal” or “broad range” immunogenic helpert cell epitope linked, preferably covalently, to a B cell epitopeportion such as the one found in the carboxyl terminal portion of humanCETP involved in a neutral lipid binding or transfer activity of CETP.In order to enhance the immunogenicity of the functional site on humanCETP, i.e., the carboxyl terminal amino acids of human CETP (SEQ IDNOS96 and 97) and peptide analogues thereof linked to the antigenicpeptides comprising artificial Th epitopes from Table 1 and CETP targetantigen peptides were synthesized. These peptides antigens, shown inTable 8, are used to elicit antibodies to CETP in small animals,primates and humans. Anti-CETP antibodies control CETP activity.Thereby, the CETP antigenic peptides are expected to cause reducedaccumulation in plasma of LDL-associated cholesterol and provideprotection from and treatment of arteriosclerosis and coronary heartdisease.

EXAMPLE 10 Peptide Composition as HIV Vaccine Component for theElicitation of Neutralizing Antibodies against HIV

Progress in the development of an effective vaccine for HIV-1 has beengauged in large part by measuring the ability to induce measurablevirus-specific CD8⁺ cytotoxic T lymphocytes (CTLs) and neutralizingantibodies, the leading indicators of a protective immune response. Astrong correlation was observed between protection against infection andlevels of neutralizing Abs in nonhuman primates infected with HIV-1 orsimian HIV (SHIV). However, little progress has been made in generatingneutralizing antibodies over the past decades. Part of the problem isthe weak neutralizing antibody response generated by candidate HIV-1vaccines. A recent article entitled “Toward an HIV Type 1 vaccine thatgenerates potent, broadly cross-reactive neutralizing antibodies” byMontefiori et al (AIDS Res and Hum Retrovir, 1999, 15:689-698) providesa state-of-the-art review of the difficulties encountered in HIVvaccines development.

In an effort to enhance the neutralizing antibody response against HIV,we embarked on synthetic antigen designs aimed at eliciting potentneutralizing antibodies. The synthetic antigens employ speciallydesigned artificial Th epitopes as the immune stimulatory element of theconstruct covalently linked to HIV neutralizing B cell epitopes (e.g.Korber et al, HIV Mol Immunol Database 1997, Part III Antibody BindingSites) or neutralizing mimetopes (e.g. Scala et al, J Immunol 1999, 162:6155-6161) previously recognized by either monoclonal or polyclonalantibodies.

Amongst the HIV neutralizing B cell epitopes, an artificial V3 consensussequence was designed (SEQ ID NO: 125) comprising an optimal frame ofthe V3 principal neutralizing determinant of HIV gp120 (Wang, C. Y.,U.S. Pat. No. 5,763,160). These include the most frequent amino acid foreach of the amino acid positions within that frame based on analyses ofamino acid sequences from over 1,000 HIV isolates from subtypes A to G,along with sequences derived from linear or confromational epitopes ofHIV gp120/gp41 regions shown here in Tables 9 and 10 in SEQ ID NOS;125,126-129, 148-153. The neutralizing mimetopes of Scala et al (SEQ IDNOS:130-135) are also synthesized as peptides of the invention, SEQ IDNOS: 136-147, as shown in Table 9.

The immunogenic peptide constructs of the invention shown in Table 10are wholly synthetic and were synthesized by the solid-phase methodoutlined in Example 1. These immunogenic peptide constructs furtherillustrate the utility of the artificial Th of the present invention inHIV vaccine development.

Each peptide in Table 10 can be represented by the formula(A)_(n)-(Th)_(m)-(B)_(o)-(HIV B neutralizing epitope)-X or (HIVneutralizing epitope)-(B)_(o)-(Th)_(m)-(A)_(n)-X. The HIV neutralizingepitopes include SEQ ID NOS: 125 and 130-135. The immunogenic peptidescomprise one or more Th sites derived from artificial Th (as shown inTable 1). Each peptide of this example have Gly-Gly or (ε-N)Lys spacersbetween immunogenic elements, but peptides of the invention may haveother spacers or no spacers.

Peptides of these examples may comprise an optional Invimmunostimulatory site (SEQ ID NO: 27). It is understood however thatthe invention is not limited to the use of Inv as an additionalimmunostimulatory element.

Representative peptide constructs of the invention, as listed in Table10 (SEQ ID NOS: 148-153) were synthesized, cleaved, cyclized andpurified as described in Example 1. Each peptide construct wasformulated for immunization into small animals such as guinea pigs, orinto larger animals such as pigs or baboons for evaluation of itsimmunogenicity. Each of the peptides was suspended in a volume of 0.5 mLcontaining representative emulsifiers or adjuvants such as ISA51,ISA720, DDA or monophosphoryl lipid A (MPL). the dose was 100 μg ofpeptide for guinea pig or 300 μg or peptide for swine or baboons and theanimals were immunized intramuscularly.

Animals received injections on weeks 0, 3 and 6 as specified in Table10. Test bleeds at 5, 8, 10 and 11 weeks post initial immunization wereevaluated for reactivities with target epitopes by B cell epitopepeptide ELISA as described in Example 1, and further tested for theirability to neutralize HIV-1 as described in details in Example 5.

All peptides tested elicited strong side-directed cross reactivities tothe corresponding target peptide, as shown by Log₁₀ titers on the anti-Bepitope peptide ELISAs of greater than 4. Neutralization of HIV-1 wasalso observed for immune sera obtained from guinea pigs, and baboons.This functional reactivity by the baboon sera is noteworthy insomuch asthe neutralization of human HIV by the baboon sera is nearly a humansystem. The results are strong indicators of the efficacy of a peptideconstruct of the invention as an agent for the prevention and/orimmunotherapy of HIV infection by active immunization.

TABLE 1 Model, Prototype, and Artificial Idealized Th Epitopes ThIdentifier Amino Acid Sequence a. MVF Th and Th epitopes derivedtherefrom MVF Th SEQ ID NO:1   LSEIKGVIVHRLEGV SSAL1 Thl SEQ ID NO:2 DLSDLKGL LL HKLDGL SEQ ID NO:3  EI EIR I II RIE I SEQ ID NO:4   V  V  VVV  V  V SEQ ID NO:5   F  F  F FF  F  F SEQ ID NO:6   ISEIKGVIVHKIEGISEQ ID NO:7   MT  RT   TRM TM SEQ ID NO:8   L          L  V SEQ ID NO:6  ISEIKGVIVHKIEGI SEQ ID NO:9    T  RT   TR  T SEQ ID NO:10  MSEIKGVIVHKLEGM SEQ ID NO:11   LT MRT   TRM TV SEQ ID NO:6  ISEIKGVIVHKIEGI SEQ ID NO:12   ITEIRTVIVTRIETI SEQ ID NO:13  MSEMKGVIVHKMEGM SEQ ID NO:14   LTEIRTVIVTRLETV SEQ ID NO:15ISISEIKGVIVHKIEGILF SEQ ID NO:16   MT  RT   TRM TM SEQ ID NO:17  L          L  V SEQ ID NO:15 ISISEIKGVIVHKIEGILF SEQ ID NO:18   T  RT   TR  T SEQ ID NO:19 ISLSEIKGVIVHKLEGMLF SEQ ID NO:105  MT MRT   TRM TV SEQ ID NO:123 ISLTEIRTVIVTRLETVLF SEQ ID NO:124   I         I  I SEQ ID NO:15 ISISEIKGVIVHKIEGILF SEQ ID NO:20ISITEIRTVIVTRIETILF SEQ ID NO:21 ISMSEMKGVIVHKMEGMLF SEQ ID NO:22ISLTEIRTVIVTRLETVLF b. HBsAg Th, Prototype and Derivatives HbsAg.Th SEQID NO:23    FFLLTRILTIPQSLD SEQ ID NO:24 KKKFFLLTRILTIPQSLD SEQ ID NO:25   FFLLTRILTIPQSL SSAL2 Th2 SEQ ID NO:26 KKKLF LL TK L LTLPQSLD SEQ IDNO 27 RRRIK II  R I I I L IR SEQ ID NO:28    VR VV    V V V I V SEQ IDNO:29    F FF    F F F V F SEQ ID NO:30             F SEQ ID NO:31KKKIITITRIITIITTID SEQ ID NO:32 KKKIITITRIITIITTI SEQ ID NO:33KKKMMTMTRMITMITTID SEQ ID NO:34    FITMDTKFLLASTMIL SEQ ID NO:35KKKFITMDTKFLLASTHIL

TABLE 2 Immunogenicity of LHRH Peptides SEQ ID Description of AntigenicFormulations NO: Peptide no. castrated no. castrated a. MVF ThDerivatives 36 (SEQ ID NO:1)-GG-(LHRH)^(a) 400 μg/dose 8/10 400 μg/dose5/5 IFA (0,3,6wpi) Alum (0,3,6wpi) 37 (SEQ ID NO:2)-GG-(LHRH)^(a) 400μg/dose 9/10 400 μg/dose 2/5 38 (SEQ ID NO:3)-GG-(LHRH)^(a) IFA(0,3,6wpi) Alum (0,3,6wpi) 39 (SEQ ID NO:4)-GG-(LHRH)^(a) 40 (SEQ IDNO:5)-GG-(LHRH)^(a) 41 (SEQ ID NO:6)-GG-(LHRH)^(a) 400 μg/dose 6/6 25μg/dose 3/8 42 (SEQ ID NO:7)-GG-(LHRH)^(a) IFA (0,3,6wpi) Alum(0,3,6wpi) 43 (SEQ ID NO:8)-GG-(LHRH)^(a) 41 (SEQ ID NO:6)-GG-(LHRH)^(a)N.D. 25 μg/dose 1/6 44 (SEQ ID NO:9)-GG-(LHRH)^(a) Alum (0,3,6wpi) 45(Inv) -GG-(SEQ ID NO:6)-GG- N.D. 25 μg/dose 5/5 (LHRH)^(a) Alum (0,3wpi) 46 (Inv)^(b)-GG-(SEQ ID NO:9)-GG- (LHRH)^(a) 47 (SEQ IDNO:10)-GG-(LHRH)^(a) 100 μg/dose 4/6 25 μg/dose 1/8 48 (SEQ IDNO:11)-GG-(LHRH)^(a) IFA (0,3,6wpi) Alum (0,3 wpi) 45 (Inv)^(b)-GG-(SEQID NO:6)- N.D. 25 #g/dose 2/6 GG-(LHRH)^(a) Alum (0,3 wpi) 49 (SEQ IDNO:12)-GG-(LHRH)^(a) N.D. 25 μg/dose 5/6 Alum (0,3 wpi) 50 (SEQ IDNO:13)-GG-(LHRH)^(a) N.D. 25 μg/dose 0/6 Alum (0,3 wpi) 51 (SEQ IDNO:14)-GG-(LHRH)^(a) N.D. 25 μg/dose 5/6 Alum (0,3 wpi) 52(Inv)^(b)-GG-(SEQ ID NO:14)- N.D. 25 μg/dose 3/6 GG-(LHRH)^(a) Alum (0,3wpi) 53 (SEQ ID NO:15)-GG-(LHRH)^(a) N.D. 25 μg/dose 4/8 54 (SEQ IDNO:16)-GG-(LHRH)^(a) Alum (0,3 wpi) 55 (SEQ ID NO:17)-GG-(LHRH)^(a) 53(SEQ ID NO:15)-GG-(LHRH)^(a) N.D. 25 μg/dose 6/6 56 (SEQ IDNO:18)-GG-(LHRH)^(a) Alum (0,3 wpi) 57 (Inv)^(b)-GG-(SEQ ID NO:15)- N.D.25 μg/dose 6/6 GG-(LHRH)^(a) Alum (0,3 wpi) 6/6 58 (Inv)^(b)-GG-(SEQ IDNO:18)- GG-(LHRH)^(a) 59 (SEQ ID NO:19)-GG-(LHRH)^(a) 100 μg/dose 6/6 25μg/dose 14/14 106 (SEQ ID NO:105)-GG-(LHRH)^(a) IFA (0,3,6wpi) Alum (0,3wpi) 53 (SEQ ID NO:15)-GG-(LHRH)^(a) N.D. 25 μg/dose 1/6 Alum (0,3 wpi)60 (Inv)^(b)-(SEQ ID NO:15)-GG- N.D. 25 μg/dose 4/6 (LHRH)^(a) Alum (0,3wpi) 61 (SEQ ID NO:2O)-GG-(LHRH)^(a) N.D. 25 μg/dose 4/6 Alum (0,3 wpi)62 (Inv)^(b)-GG-(SEQ ID NO:20)- N.D. 25 μg/dose 2/6 GG-(LHRH)^(a) Alum(0,3 wpi) 63 (Inv)-(SEQ ID NO:21)-GG- N.D. 25 μg/dose 1/6 (LHRH)^(a)Alum (0,3 Wpi) 64 (SEQ ID NO:22)-GG-(LHRH)^(a) N.D. 25 μg/dose 4/6 Alum(0,3 wpi) b. HBSAg Th Derivatives 65 (SEQ ID NO:23)-GG-(LHRH)^(a) 400μg/dose 10/10 400 μg/dose 0/5 IFA (0,3,6wpi) Alum (0.3,6 wpi) 66 (SEQ IDNO:26)-GG-(LHRH)^(a) 400 μg/dose 9/10 400 μg/dose 2/5 67 (SEQ IDNo:27)-GG-(LHRH)^(a) IFA (0,3,6spi) Alum (0,3,6 wpi) 68 (SEQ IDNO:28)-GG-(LHRH)^(a) 69 (SEQ ID NO:29)-GG-(LHRH)^(a) 70 (SEQ IDNO:30)-GG-(LHRH)^(a) 71 (SEQ ID NO:31)-GG-(LHRH)^(a) N.D. 25 μg/doseAlum 8/8 (0,3 wpi) 72 (SEQ ID NO:32)-GG-(LHRH)^(a) N.D. 25 μg/dose Alum4/6 (0,3 wpi) 73 (SEQ ID NO:33)-GG-(LHRH)^(a) 100 μg/dose 4/6 25 μg/doseAlum 0/6 IFA (0,3,6wpi) (0,3 wpi) 74 (SEQ ID NO:34)-GG-(LHRH)^(a) 100μg/dose 6/6 25 μg/dose 5/8 IFA (0,3,6wpi) Alum (0,3 wpi) 75(Inv)^(b)-GG-(SEQ ID NO:34)- N.D. 25 μg/dose 4/6 GG-(LHRH)^(a) Alum (0.3wpi) 76 (SEQ ID ND:35)-GG-(LHRH)^(a) N.D. 25 μg/dose 0/8 Alum (0,3 wpi)KLH^(c)-(LHRH)^(a) N.D. 50 μg/dose Alum 2/8 (0,3 wpi) ^(a)LHRH =EHWSYGLRPG (SEQ ID NO:77) ^(b)INV = Invasin domain (SEQ ID NO:78)^(c)KLH = Keyhole limpet hemocyanin ^(d)Hinge spacer = PPXPXP (SEQ IDNO:79)

TABLE 3 Evaluation of Antibody Specificity for the Target Antigenic SiteSEQ ID NO: LHRH Reactivity^(a) Th Reactivity^(b) 53 and 56 6/6 0/6^(c)71 8/8 0/8^(d) 74 4/8 0/8^(e) ^(a)Number of animals with anti-LHRHtiters > 1:1000/total animals immunized. The ELISA peptide was SEQ IDNO:77. ^(b)Number of animals with anti-Th reactivity > 0.100 A₄₉₀/totalanimals immunized. Sera were diluted 1:100 and all A₄₉₀ values were atbackground values for the respective Th peptides. ^(c)ELISA peptide wasa mixture of two peptides: SEQ ID NOS:15 and 18. ^(d)ELISA peptide wasSEQ ID NO:31. ^(e)ELISA peptide was SEQ ID NO:34.

TABLE 4 Evaluation of Artificial Th/LHRH Peptide Compositions IncludingMixture 0 5 8 10 14 18 22 Immunogen^(a) wpi wpi wpi wpi wpi wpi wpi SEQID NO: 64 + 0/8^(b) 3/8 8/8 8/8 7/8 5/8 3/8 Alum SEQ ID NO: 57 and 0/66/6 6/6 6/6 6/6 6/6 6/6 58 + Alum SEQ ID NO: 64 + 0/6 6/6 6/6 6/6 6/66/6 6/6 SEQ ID NO: 57 and 58 + Alum ^(a)Individual LHRH peptidecompositions or the mixed LHRH peptide composition were formulated onalum. Immunization schedule: 25 μg/dose at 0 and 3 wpi. ^(b)Number ofanimals immunocastrated/total number of animals in group. Animals werescored as immunocastrated when serum testosterone values were <0.1nmol/L to undetectable.

TABLE 5 Immunogenicity of Somatostatin Antigenic Peptides ImmunogenicityLog₁₀ Description of Responding ELISA SEQ ID NO: Antigenic PeptideAdjuvant WPI^(b) n = 4 Titer^(c) 80 Somatostatin^(a) 0.4% Alum 6 0 — (0,2, 4 WPI) 8 0 — 81, 82, 83 (SEQ ID NOS: 6, 7, 8) -GG- 0.4% Alum 5 1/42.38 (Somatostatin) (0, 3, 6 WPI) 8 4/4 3.33 84, 85, 86 (Somatostatin)-GG- 0.4% Alum 5 3/4 3.24 (SEQ ID NOS: 6, 7, 8) (0, 3, 6 WPI) 8 4/4 3.1287 (SEQ ID NO: 31) -GG- 0.4% Alum WPI) 8 4/4 3.12 Somatostatin (0, 3, 6WPI) ^(a)Sequence of somatostatin: AGCKNFFWKTFTSC (SEQ ID NO: 80)^(b)WPI = Weeks post-immunization ^(c)Test results for pooled sera fromELISA-reactive animals.

TABLE 6 Immunogenicity of IgE-CH3 Antigenic Peptides ImmunogenicityDescription of No. Log₁₀ SEQ ID NO: Antigenic Peptide respondingELISA^(b) 93, 94 (SEQ ID NOS: 15, 18)-GG- 3/3 3.30 (SEQ ID NO: 92)^(a)95, 96, 97 (SEQ ID NOS: 6, 7, 8)-GG- 3/3 3.32 (SEQ ID NO: 92)^(a) 98(SEQ ID NO: 31)-GG- 3/3 2.35 (SEQ ID NO: 92)^(a) 99 Inv^(c)-GG-(SEQ IDNO: 31)-GG- 3/3 3.28 (SEQ ID NO: 92)^(a) KLH^(d)-(SEQ ID NO: 92)^(a) 2/20.49 ^(a)Modified IgE-CH3 site (SEQ ID NO: 92) ^(b)Average for animalsof the group ^(c)Invasin domain peptide (SEQ ID NO: 78) ^(d)KeyholeLimpet Hemocyanin

TABLE 7 Artificial Th and SSAL Taget Antigenic Peptides for ImprovedImmunogenicity and Breadth of FMDV Neutralization Log₁₀ No. of Anti-Log₁₀ of FMDV (MPD₅₀) Animals FMDVP1^(d) Neutralized by Serum^(d,c) SEQResponding ELISA A₁₂ O-1 O-1 ID NO: Target Antigenic Peptide WPI (n = 3)Titer FP (JH) A-FL P2 A-23 Asia 1 A 1 101 (SEQ ID NO:31)-GG-O_(complete) 5 3 5.158 3.0 4.5 2.0 5.0 _(SSAL)[134-158(T→C)^(a)-169], 10 3 2.5 4.5 6.0 cyclized 102 Inv-GG-(SEQ ID NO: 5 35.256 3.0 4.5 2.0 4.5 31)-GG-Asia _(SSAL)[134 10 3 1.0 3.0 2.5(T→C)^(b)-158 (R→C)^(c)-169], cyclized ^(a)T₁₅₈ of the native sequencewas replaced by C. ^(b)T₁₃₄ of native sequence was replaced by C.^(c)R₁₅₈ of the native sequence was replaced by C. ^(d)Reactivities ofpooled sera from ELISA-reactive animals. ^(e)Serum for neutralizationassays diluted 1:100.

TABLE 8 CETP ANTIGENIC PEPTIDES SEQ ID NO DESCRIPTION OF ANTIGENICPEPTIDE 110,111 (SEQ ID NOS:15,18)-□NLys-(SEQ ID NO:106) 112,113 (SEQ IDNOS:15,18)-□NLys-(SEQ ID NO:107) 114 (SEQ ID NO:31)-□NLys-(SEQ IDNO:107) 115 (SEQ ID NO:22)-□NLys-(SEQ ID NO:107) 116,117 (SEQ IDNO:15,18)-□NLys-(SEQ ID NO:108)^(a) 118,119 (SEQ ID NO:15,18)-□NLys-(SEQID NO:109)^(b) Note: ^(a)= Phe Gly Phe Pro Lys His Leu Leu Val Asp Phe    Leu Gln Ser Leu Ser (SEQ ID NO:108) ^(b)= Leu Asp Gly Cys Leu LeuLeu Gln Met Asp Phe    Gly Phe Pro Lys His Leu Leu Val Asp Phe Leu   Gln Ser Leu Ser (SEQ ID NO:109)

TABLE 9 Amino Acid Sequence of HIV Pep- Neutralizing EpitopesDescription of Th tide Amino Acid Sequences Of Representative (SEQ IDNO) Epitopes Code Peptide Constructs ESVEINCTRPNNNTRKSIRIGPGQAFYATGDSimplified MVF Th lib p2846b ISISEIKGVIVHKIEGILF-(εN)K- (SEQ ID NO:125)(SEQ ID No:15, 18)    T  RT   TR  T ESVEINCTRPNNNTRKSIRIGPGQAFYATGD (SEQID NO:126, 127) Simplified [I, L] MVF Th p2868bISLTEIRTVIVTRLETVLF--(εN)K- (SEQ ID No:110, 111)   I          I  IESVEINCTRPNNNTRKSIRIGPGQAFYATGD (SEQ ID NO:128, 129) KSSGKLISLSimplified MVF Th p2942 KSSGKLISL-(εN)K-ISISEIKGVIVHKIEGILF (SEQ IDNO:130) (SEQ ID No:15,18)                    T  RT   TR  T (SEQ IDNO:136,137) CNGRLYCGP Simplified MVF Th p2937CNGRLYCGP-(εN)K-ISISEIKGVIVHKIEGILF (SEQ ID NO:131) (SEQ ID No:15,18)                   T  RT   TR  T (SEQ ID NO:138, 139) (C)GTKLVCFAASimplified MVF Th P2939 (C)GTKLVCFAA-(εN)K-ISISEIKGVIVHKIEGILF (SEQ IDNO:132) (SEQ ID No:15, 18)                       T  RT   TR  T (SEQ IDNO:140,141) KRIVIGPQT Simplified MVF Th p2944KRIVIGPQT-(εN)K-ISISEIKGVIVHKIEGILF (SEQ ID NO:133) (SEQ ID NO:15, 18)                   T  RT   TR  T (SEQ ID NO:142,143) CAGGLTCSVSimplified MVF Th p2940 CAGGLTCSV-(εN)K-ISISEIKGVIVHKIEGILF (SEQ IDNO:134) (SEQ ID No:15, 18)                    T  RT   TR  T (SEQ IDNO:144, 145) (C)SGRLYCHESW Simplified MVF Th p2941(C)SGRLYCHESW-(εN)K-ISISEIKGVIVHKIEGILF (SEQ ID NO:135) (SEQ ID No:15,18)                        T  RT   TR  T (SEQ ID No:146, 147)

TABLE 10 Immunogenicity of Representative Peptides of the InventionSerum Titer by Log₁₀ Neutralization ELISA Assay Description of Titer on(HIV-1 MN/H9) Adjuvant and Peptide Representative Target 50% 90% SpeciesGroup # Immunization Schedule Code Peptide Constructs WPI AntigenInhibit. Inhibit. Guinea Pig 1 Oil in water emulsion p2846 b IS(1,4,9PALINDROMIC 5 4.461 3972 425 (0, 3, 6 WPI) (Seq ID Nos. Th simplifiedlib) LF-GG-“V3” 8 4.383 10276 535 148, 149) All Concensus 10 4.245 42451006 2 Oil in water emulsion p2868 lib(1, L) (εN)K - “V3” 5 4.489 1198206 (0, 3, 6 WPI) (Seq ID Nos. All Concensus 8 4.291 3295 1092 150, 151)10 4.239 4233 1147 Baboon 1 Oil in water emulsion p2868 lib(1, L)(εN)K - “V3” 8 4.375 948 152 (0, 3, 6 WPI) (Seq ID Nos. All Concensus 11over 23745 2811 150, 151)

151 15 amino acids amino acid linear peptide 1 Leu Ser Glu Ile Lys GlyVal Ile Val His Arg Leu 1 5 10 Glu Gly Val 15 16 amino acids amino acidlinear peptide 2 Asp Leu Ser Asp Leu Lys Gly Leu Leu Leu His Lys 1 5 10Leu Asp Gly Leu 15 16 amino acids amino acid LINEAR peptide 3 Glu IleSer Glu Ile Arg Gly Ile Ile Ile His Arg 1 5 10 Ile Glu Gly Ile 15 16amino acids amino acid LINEAR peptide 4 Asp Val Ser Asp Val Lys Gly ValVal Val His Lys 1 5 10 Val Asp Gly Val 15 16 amino acids amino acidLINEAR peptide 5 Asp Phe Ser Asp Phe Lys Gly Phe Phe Phe His Lys 1 5 10Phe Asp Gly Phe 15 15 amino acids amino acid linear peptide 6 Ile SerGlu Ile Lys Gly Val Ile Val His Lys Ile 1 5 10 Glu Gly Ile 15 15 aminoacids amino acid linear peptide 7 Met Thr Glu Ile Arg Thr Val Ile ValThr Arg Met 1 5 10 Glu Thr Met 15 15 amino acids amino acid linearpeptide 8 Leu Ser Glu Ile Lys Gly Val Ile Val His Lys 1 5 10 Leu Glu GlyVal 15 15 amino acids amino acid linear peptide 9 Ile Thr Glu Ile ArgThr Val Ile Val Thr Arg Ile 1 5 10 Glu Thr Ile 15 15 amino acids aminoacid linear peptide 10 Met Ser Glu Ile Lys Gly Val Ile Val His Lys Leu 15 10 Glu Gly Met 15 15 amino acids amino acid linear peptide 11 Leu ThrGlu Met Arg Thr Val Ile Val Thr Arg Met 1 5 10 Glu Thr Val 15 15 aminoacids amino acid linear peptide 12 Ile Thr Glu Ile Arg Thr Val Ile ValThr Arg Ile 1 5 10 Glu Thr Ile 15 15 amino acids amino acid linearpeptide 13 Met Ser Glu Met Lys Gly Val Ile Val His Lys Met 1 5 10 GluGly Met 15 15 amino acids amino acid linear peptide 14 Leu Thr Glu IleArg Thr Val Ile Val Thr Arg Leu 1 5 10 Glu Thr Val 15 19 amino acidsamino acid linear peptide 15 Ile Ser Ile Ser Glu Ile Lys Gly Val Ile ValHis 1 5 10 Lys Ile Glu Gly Ile Leu Phe 15 19 amino acids amino acidlinear peptide 16 Ile Ser Met Thr Glu Ile Arg Thr Val Ile Val Thr 1 5 10Arg Met Glu Thr Met Leu Phe 15 19 amino acids amino acid linear peptide17 Ile Ser Leu Ser Glu Ile Lys Gly Val Ile Val His 1 5 10 Lys Leu GluGly Val Leu Phe 15 19 amino acids amino acid linear peptide 18 Ile SerIle Thr Glu Ile Arg Thr Val Ile Val Thr 1 5 10 Arg Ile Glu Thr Ile LeuPhe 15 19 amino acids amino acid linear peptide 19 Ile Ser Leu Ser GluIle Lys Gly Val Ile Val His 1 5 10 Lys Leu Glu Gly Met Leu Phe 15 19amino acids amino acid linear peptide 20 Ile Ser Ile Thr Glu Ile Arg ThrVal Ile Val Thr 1 5 10 Arg Ile Glu Thr Ile Leu Phe 15 19 amino acidsamino acid linear peptide 21 Ile Ser Met Ser Glu Met Lys Gly Val Ile ValHis 1 5 10 Lys Met Glu Gly Met Leu Phe 15 19 amino acids amino acidlinear peptide 22 Ile Ser Leu Thr Glu Ile Arg Thr Val Ile Val Thr 1 5 10Arg Leu Glu Thr Val Leu Phe 15 15 amino acids amino acid linear peptide23 Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln 1 5 10 Ser Leu Asp 1518 amino acids amino acid linear peptide 24 Lys Lys Lys Phe Phe Leu LeuThr Arg Ile Leu Thr 1 5 10 Ile Pro Gln Ser Leu Asp 15 14 amino acidsamino acid linear peptide 25 Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile ProGln 1 5 10 Ser Leu 18 amino acids amino acid linear peptide 26 Lys LysLys Leu Phe Leu Leu Thr Lys Leu Leu Thr 1 5 10 Leu Pro Gln Ser Leu Asp15 18 amino acids amino acid linear peptide 27 Arg Arg Arg Ile Lys IleIle Thr Arg Ile Ile Thr 1 5 10 Ile Pro Leu Ser Ile Arg 15 18 amino acidsamino acid linear peptide 28 Lys Lys Lys Val Arg Val Val Thr Lys Val ValThr 1 5 10 Val Pro Ile Ser Val Asp 15 18 amino acids amino acid linearpeptide 29 Lys Lys Lys Phe Phe Phe Phe Thr Lys Phe Phe Thr 1 5 10 PhePro Val Ser Phe Asp 15 18 amino acids amino acid linear peptide 30 LysLys Lys Leu Phe Leu Leu Thr Lys Leu Leu Thr 1 5 10 Leu Pro Phe Ser LeuAsp 15 18 amino acids amino acid linear peptide 31 Lys Lys Lys Ile IleThr Ile Thr Arg Ile Ile Thr 1 5 10 Ile Ile Thr Thr Ile Asp 15 17 aminoacids amino acid linear peptide 32 Lys Lys Lys Ile Ile Thr Ile Thr ArgIle Ile Thr 1 5 10 Ile Ile Thr Thr Ile 15 18 amino acids amino acidlinear peptide 33 Lys Lys Lys Met Met Thr Met Thr Arg Met Ile Thr 1 5 10Met Ile Thr Thr Ile Asp 15 16 amino acids amino acid linear peptide 34Phe Ile Thr Met Asp Thr Lys Phe Leu Leu Ala Ser 1 5 10 Thr His Ile Leu15 19 amino acids amino acid linear peptide 35 Lys Lys Lys Phe Ile ThrMet Asp Thr Lys Phe Leu 1 5 10 Leu Ala Ser Thr His Ile Leu 15 27 aminoacids amino acid linear peptide 36 Leu Ser Glu Ile Lys Gly Val Ile ValHis Arg Leu 1 5 10 Glu Gly Val Gly Gly Glu His Trp Ser Tyr Gly Leu 15 20Arg Pro Gly 25 28 amino acids amino acid linear peptide 37 Asp Leu SerAsp Leu Lys Gly Leu Leu Leu His Lys 1 5 10 Leu Asp Gly Leu Gly Gly GluHis Trp Ser Tyr Gly 15 20 Leu Arg Pro Gly 25 28 amino acids amino acidlinear peptide 38 Glu Ile Ser Glu Ile Arg Gly Ile Ile Ile His Arg 1 5 10Ile Glu Gly Ile Gly Gly Glu His Trp Ser Tyr Gly 15 20 Leu Arg Pro Gly 2528 amino acids amino acid linear peptide 39 Asp Val Ser Asp Val Lys GlyVal Val Val His Lys 1 5 10 Val Asp Gly Val Gly Gly Glu His Trp Ser TyrGly 15 20 Leu Arg Pro Gly 25 28 amino acids amino acid linear peptide 40Asp Phe Ser Asp Phe Lys Gly Phe Phe Phe His Lys 1 5 10 Phe Asp Gly PheGly Gly Glu His Trp Ser Tyr Gly 15 20 Leu Arg Pro Gly 25 27 amino acidsamino acid linear peptide 41 Ile Ser Glu Ile Lys Gly Val Ile Val His LysIle 1 5 10 Glu Gly Ile Gly Gly Glu His Trp Ser Tyr Gly Leu 15 20 Arg ProGly 25 27 amino acids amino acid linear peptide 42 Met Thr Glu Ile ArgThr Val Ile Val Thr Arg Met 1 5 10 Glu Thr Met Gly Gly Glu His Trp SerTyr Gly Leu 15 20 Arg Pro Gly 25 27 amino acids amino acid linearpeptide 43 Leu Ser Glu Ile Lys Gly val Ile Val His Lys Leu 1 5 10 GluGly Val Gly Gly Glu His Trp Ser Tyr Gly Leu 15 20 Arg Pro Gly 25 27amino acids amino acid linear peptide 44 Ile Thr Glu Ile Arg Thr Val IleVal Thr Arg Ile 1 5 10 Glu Thr Ile Gly Gly Glu His Trp Ser Tyr Gly Leu15 20 Arg Pro Gly 25 45 amino acids amino acid linear peptide 45 Thr AlaLys Ser Lys Lys Phe Pro Ser Tyr Thr Ala 1 5 10 Thr Tyr Gln Phe Gly GlyIle Ser Glu Ile Lys Gly 15 20 Val Ile Val His Lys Ile Glu Gly Ile GlyGly Glu 25 30 35 His Trp Ser Tyr Gly Leu Arg Pro Gly 40 45 45 aminoacids amino acid linear peptide 46 Thr Ala Lys Ser Lys Lys Phe Pro SerTyr Thr Ala 1 5 10 Thr Tyr Gln Phe Gly Gly Ile Thr Glu Ile Arg Thr 15 20Val Ile Val Thr Arg Ile Glu Thr Ile Gly Gly Glu 25 30 35 His Trp Ser TyrGly Leu Arg Pro Gly 40 45 27 amino acids amino acid linear peptide 47Met Ser Glu Ile Lys Gly Val Ile Val His Lys Leu 1 5 10 Glu Gly Met GlyGly Glu His Trp Ser Tyr Gly Leu 15 20 Arg Pro Gly 25 27 amino acidsamino acid linear peptide 48 Leu Thr Glu Met Arg Thr Val Ile Val Thr ArgMet 1 5 10 Glu Thr Val Gly Gly Glu His Trp Ser Tyr Gly Leu 15 20 Arg ProGly 25 27 amino acids amino acid linear peptide 49 Ile Thr Glu Ile ArgThr Val Ile Val Thr Arg Ile 1 5 10 Glu Thr Ile Gly Gly Glu His Trp SerTyr Gly Leu 15 20 Arg Pro Gly 25 27 amino acids amino acid linearpeptide 50 Met Ser Glu Met Lys Gly Val Ile Val His Lys Met 1 5 10 GluGly Met Gly Gly Glu His Trp Ser Tyr Gly Leu 15 20 Arg Pro Gly 25 27amino acids amino acid linear peptide 51 Leu Thr Glu Ile Arg Thr Val IleVal Thr Arg Leu 1 5 10 Glu Thr Val Gly Gly Glu His Trp Ser Tyr Gly Leu15 20 Arg Pro Gly 25 45 amino acids amino acid linear peptide 52 Thr AlaLys Ser Lys Lys Phe Pro Ser Tyr Thr Ala 1 5 10 Thr Tyr Gln Phe Gly GlyLeu Thr Glu Ile Arg Thr 15 20 Val Ile Val Thr Arg Leu Glu Thr Val GlyGly Glu 25 30 35 His Trp Ser Tyr Gly Leu Arg Pro Gly 40 45 31 aminoacids amino acid linear peptide 53 Ile Ser Ile Ser Glu Ile Lys Gly ValIle Val His 1 5 10 Lys Ile Glu Gly Ile Leu Phe Gly Gly Glu His Trp 15 20Ser Tyr Gly Leu Arg Pro Gly 25 30 31 amino acids amino acid linearpeptide 54 Ile Ser Met Thr Glu Ile Arg Thr Val Ile Val Thr 1 5 10 ArgMet Glu Thr Met Leu Phe Gly Gly Glu His Trp 15 20 Ser Tyr Gly Leu ArgPro Gly 25 30 31 amino acids amino acid linear peptide 55 Ile Ser LeuSer Glu Ile Lys Gly Val Ile Val His 1 5 10 Lys Leu Glu Gly Val Leu PheGly Gly Glu His Trp 15 20 Ser Tyr Gly Leu Arg Pro Gly 25 30 31 aminoacids amino acid linear peptide 56 Ile Ser Ile Thr Glu Ile Arg Thr ValIle Val Thr 1 5 10 Arg Ile Glu Thr Ile Leu Phe Gly Gly Glu His Trp 15 20Ser Tyr Gly Leu Arg Pro Gly 25 30 49 amino acids amino acid linearpeptide 57 Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala 1 5 10 ThrTyr Gln Phe Gly Gly Ile Ser Ile Ser Glu Ile 15 20 Lys Gly Val Ile ValHis Lys Ile Glu Gly Ile Leu 25 30 35 Phe Gly Gly Glu His Trp Ser Tyr GlyLeu Arg Pro 40 45 Gly 49 amino acids amino acid linear peptide 58 ThrAla Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala 1 5 10 Thr Tyr Gln Phe GlyGly Ile Ser Ile Thr Glu Ile 15 20 Arg Thr Val Ile Val Thr Arg Ile GluThr Ile Leu 25 30 35 Phe Gly Gly Glu His Trp Ser Tyr Gly Leu Arg Pro 4045 Gly 31 amino acids amino acid linear peptide 59 Ile Ser Leu Ser GluIle Lys Gly Val Ile Val His 1 5 10 Lys Leu Glu Gly Met Leu Phe Gly GlyGlu His Trp 15 20 Ser Tyr Gly Leu Arg Pro Gly 25 30 47 amino acids aminoacid linear peptide 60 Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala 15 10 Thr Tyr Gln Phe Ile Ser Ile Ser Glu Ile Lys Gly 15 20 Val Ile ValHis Lys Ile Glu Gly Ile Leu Phe Gly 25 30 35 Gly Glu His Trp Ser Tyr GlyLeu Arg Pro Gly 40 45 31 amino acids amino acid linear peptide 61 IleSer Ile Thr Glu Ile Arg Thr Val Ile Val Thr 1 5 10 Arg Ile Glu Thr IleLeu Phe Gly Gly Glu His Trp 15 20 Ser Tyr Gly Leu Arg Pro Gly 25 30 49amino acids amino acid linear peptide 62 Thr Ala Lys Ser Lys Lys Phe ProSer Tyr Thr Ala 1 5 10 Thr Tyr Gln Phe Gly Gly Ile Ser Ile Thr Glu Ile15 20 Arg Thr Val Ile Val Thr Arg Ile Glu Thr Ile Leu 25 30 35 Phe GlyGly Glu His Trp Ser Tyr Gly Leu Arg Pro 40 45 Gly 47 amino acids aminoacid linear peptide 63 Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala 15 10 Thr Tyr Gln Phe Ile Ser Met Ser Glu Met Lys Gly 15 20 Val Ile ValHis Lys Met Glu Gly Met Leu Phe Gly 25 30 35 Gly Glu His Trp Ser Tyr GlyLeu Arg Pro Gly 40 45 31 amino acids amino acid linear peptide 64 IleSer Leu Thr Glu Ile Arg Thr Val Ile Val Thr 1 5 10 Arg Leu Glu Thr ValLeu Phe Gly Gly Glu His Trp 15 20 Ser Tyr Gly Leu Arg Pro Gly 25 30 27amino acids amino acid linear peptide 65 Phe Phe Leu Leu Thr Arg Ile LeuThr Ile Pro Gln 1 5 10 Ser Leu Asp Gly Gly Glu His Trp Ser Tyr Gly Leu15 20 Arg Pro Gly 25 30 amino acids amino acid linear peptide 66 Lys LysLys Leu Phe Leu Leu Thr Lys Leu Leu Thr 1 5 10 Leu Pro Gln Ser Leu AspGly Gly Glu His Trp Ser 15 20 Tyr Gly Leu Arg Pro Gly 25 30 30 aminoacids amino acid linear peptide 67 Arg Arg Arg Ile Lys Ile Ile Thr ArgIle Ile Thr 1 5 10 Ile Pro Leu Ser Ile Arg Gly Gly Glu His Trp Ser 15 20Tyr Gly Leu Arg Pro Gly 25 30 30 amino acids amino acid linear peptide68 Lys Lys Lys Val Arg Val Val Thr Lys Val Val Thr 1 5 10 Val Pro IleSer Val Asp Gly Gly Glu His Trp Ser 15 20 Tyr Gly Leu Arg Pro Gly 25 3030 amino acids amino acid linear peptide 69 Lys Lys Lys Phe Phe Phe PheThr Lys Phe Phe Thr 1 5 10 Phe Pro Val Ser Phe Asp Gly Gly Glu His TrpSer 15 20 Tyr Gly Leu Arg Pro Gly 25 30 30 amino acids amino acid linearpeptide 70 Lys Lys Lys Leu Phe Leu Leu Thr Lys Leu Leu Thr 1 5 10 LeuPro Phe Ser Leu Asp Gly Gly Glu His Trp Ser 15 20 Tyr Gly Leu Arg ProGly 25 30 30 amino acids amino acid linear peptide 71 Lys Lys Lys IleIle Thr Ile Thr Arg Ile Ile Thr 1 5 10 Ile Ile Thr Thr Ile Asp Gly GlyGlu His Trp Ser 15 20 Tyr Gly Leu Arg Pro Gly 25 30 29 amino acids aminoacid linear peptide 72 Lys Lys Lys Ile Ile Thr Ile Thr Arg Ile Ile Thr 15 10 Ile Ile Thr Thr Ile Gly Gly Glu His Trp Ser Tyr 15 20 Gly Leu ArgPro Gly 25 30 amino acids amino acid linear peptide 73 Lys Lys Lys MetMet Thr Met Thr Arg Met Ile Thr 1 5 10 Met Ile Thr Thr Ile Asp Gly GlyGlu His Trp Ser 15 20 Tyr Gly Leu Arg Pro Gly 25 30 28 amino acids aminoacid linear peptide 74 Phe Ile Thr Met Asp Thr Lys Phe Leu Leu Ala Ser 15 10 Thr His Ile Leu Gly Gly Glu His Trp Ser Tyr Gly 15 20 Leu Arg ProGly 25 46 amino acids amino acid linear peptide 75 Thr Ala Lys Ser LysLys Phe Pro Ser Tyr Thr Ala 1 5 10 Thr Tyr Gln Phe Gly Gly Phe Ile ThrMet Asp Thr 15 20 Lys Phe Leu Leu Ala Ser Thr His Ile Leu Gly Gly 25 3035 Glu His Trp Ser Tyr Gly Leu Arg Pro Gly 40 45 31 amino acids aminoacid linear peptide 76 Lys Lys Lys Phe Ile Thr Met Asp Thr Lys Phe Leu 15 10 Leu Ala Ser Thr His Ile Leu Gly Gly Glu His Trp 15 20 Ser Tyr GlyLeu Arg Pro Gly 25 30 10 amino acids amino acid linear peptide 77 GluHis Trp Ser Tyr Gly Leu Arg Pro Gly 1 5 10 16 amino acids amino acidlinear peptide 78 Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala 1 5 10Thr Tyr Gln Phe 15 6 amino acids amino acid linear peptide 79 Pro ProXaa Pro Xaa Pro 1 5 14 amino acids amino acid linear peptide 80 Ala GlyCys Lys Asn Phe Phe Trp Lys Thr Phe Thr 1 5 10 Ser Cys 31 amino acidsamino acid linear peptide 81 Ile Ser Glu Ile Lys Gly Val Ile Val His LysIle 1 5 10 Glu Gly Ile Gly Gly Ala Gly Cys Lys Asn Phe Phe 15 20 Trp LysThr Phe Thr Ser Cys 25 30 31 amino acids amino acid linear peptide 82Met Thr Glu Ile Arg Thr Val Ile Val Thr Arg Met 1 5 10 Glu Thr Met GlyGly Ala Gly Cys Lys Asn Phe Phe 15 20 Trp Lys Thr Phe Thr Ser Cys 25 3031 amino acids amino acid linear peptide 83 Leu Ser Glu Ile Lys Gly ValIle Val His Lys Leu 1 5 10 Glu Gly Val Gly Gly Ala Gly Cys Lys Asn PhePhe 15 20 Trp Lys Thr Phe Thr Ser Cys 25 30 31 amino acids amino acidlinear peptide 84 Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr 1 5 10Ser Cys Gly Gly Ile Ser Glu Ile Lys Gly Val Ile 15 20 Val His Lys IleGlu Gly Ile 25 30 31 amino acids amino acid linear peptide 85 Ala GlyCys Lys Asn Phe Phe Trp Lys Thr Phe Thr 1 5 10 Ser Cys Gly Gly Met ThrGlu Ile Arg Thr Val Ile 15 20 Val Thr Arg Met Gly Thr Met 25 30 31 aminoacids amino acid linear peptide 86 Ala Gly Cys Lys Asn Phe Phe Trp LysThr Phe Thr 1 5 10 Ser Cys Gly Gly Leu Ser Glu Ile Lys Gly Val Ile 15 20Val His Lys Leu Glu Gly Val 25 30 34 amino acids amino acid linearpeptide 87 Lys Lys Lys Ile Ile Thr Ile Thr Arg Ile Ile Thr 1 5 10 IleIle Thr Thr Ile Asp Gly Gly Ala Gly Cys Lys 15 20 Asn Phe Phe Trp LysThr Phe Thr Ser Cys 25 30 30 amino acids amino acid linear peptide 88Cys Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser 1 5 10 Lys Leu Asn AspArg Ala Asp Ser Arg Arg Ser Leu 15 20 Trp Asp Gln Gly Asn Cys 25 30 47amino acids amino acid linear peptide 89 Ile Ser Glu Ile Lys Gly Val IleVal His Lys Ile 1 5 10 Glu Gly Ile Gly Gly Cys Asn Gln Gly Ser Phe Leu15 20 Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala Asp 25 30 35 Ser ArgArg Ser Leu Trp Asp Gln Gly Asn Cys 40 45 47 amino acids amino acidlinear peptide 90 Met Thr Glu Ile Arg Thr Val Ile Val Thr Arg Met 1 5 10Glu Thr Met Gly Gly Cys Asn Gln Gly Ser Phe Leu 15 20 Thr Lys Gly ProSer Lys Leu Asn Asp Arg Ala Asp 25 30 35 Ser Arg Arg Ser Leu Trp Asp GlnGly Asn Cys 40 45 47 amino acids amino acid linear peptide 91 Leu SerGlu Ile Lys Gly Val Ile Val His Lys Leu 1 5 10 Glu Gly Val Gly Gly CysAsn Gln Gly Ser Phe Leu 15 20 Thr Lys Gly Pro Ser Lys Leu Asn Asp ArgAla Asp 25 30 35 Ser Arg Arg Ser Leu Trp Asp Gln Gly Asn Cys 40 45 25amino acids amino acid linear peptide 92 Cys Gly Glu Thr Tyr Gln Ser ArgVal Thr His Pro 1 5 10 His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys15 20 Cys 25 46 amino acids amino acid linear peptide 93 Ile Ser Ile SerGlu Ile Lys Gly Val Ile Val His 1 5 10 Lys Ile Glu Gly Ile Leu Phe GlyGly Cys Gly Glu 15 20 Thr Tyr Gln Ser Arg Val Thr His Pro His Leu Pro 2530 35 Arg Ala Leu Met Arg Ser Thr Thr Lys Cys 40 45 46 amino acids aminoacid linear peptide 94 Ile Ser Ile Thr Glu Ile Arg Thr Val Ile Val Thr 15 10 Arg Ile Glu Thr Ile Leu Phe Gly Gly Cys Gly Glu 15 20 Thr Tyr GlnSer Arg Val Thr His Pro His Leu Pro 25 30 35 Arg Ala Leu Met Arg Ser ThrThr Lys Cys 40 45 42 amino acids amino acid linear peptide 95 Ile SerGlu Ile Lys Gly Val Ile Val His Lys Ile 1 5 10 Glu Gly Ile Gly Gly CysGly Glu Thr Tyr Gln Ser 15 20 Arg Val Thr His Pro His Leu Pro Arg AlaLeu Met 25 30 35 Arg Ser Thr Thr Lys Cys 40 42 amino acids amino acidlinear peptide 96 Met Thr Glu Ile Arg Thr Val Ile Val Thr Arg Met 1 5 10Glu Thr Met Gly Gly Cys Gly Glu Thr Tyr Gln Ser 15 20 Arg Val Thr HisPro His Leu Pro Arg Ala Leu Met 25 30 35 Arg Ser Thr Thr Lys Cys 40 42amino acids amino acid linear peptide 97 Leu Ser Glu Ile Lys Gly Val IleVal His Lys Leu 1 5 10 Glu Gly Val Gly Gly Cys Gly Glu Thr Tyr Gln Ser15 20 Arg Val Thr His Pro His Leu Pro Arg Ala Leu Met 25 30 35 Arg SerThr Thr Lys Cys 40 45 amino acids amino acid linear peptide 98 Lys LysLys Ile Ile Thr Ile Thr Arg Ile Ile Thr 1 5 10 Ile Ile Thr Thr Ile AspGly Gly Cys Gly Glu Thr 15 20 Tyr Gln Ser Arg Val Thr His Pro His LeuPro Arg 25 30 35 Ala Leu Met Arg Ser Thr Thr Lys Cys 40 45 63 aminoacids amino acid linear peptide 99 Thr Ala Lys Ser Lys Lys Phe Pro SerTyr Thr Ala 1 5 10 Thr Tyr Gln Phe Gly Gly Lys Lys Lys Ile Ile Thr 15 20Ile Thr Arg Ile Ile Thr Ile Ile Thr Thr Ile Asp 25 30 35 Gly Gly Cys GlyGlu Thr Tyr Gln Ser Arg Val Thr 40 45 His Pro His Leu Pro Arg Ala LeuMet Arg Ser Thr 50 55 60 Thr Lys Cys 38 amino acids amino acid linearpeptide 100 Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln 1 5 10 ThrLeu Pro Pro Ser Val Pro Asn Leu Arg Gly Asp 15 20 Leu Gln Val Leu AlaGln Lys Val Ala Arg Thr Pro 25 30 35 Cys Gly 56 amino acids amino acidlinear peptide 101 Lys Lys Lys Ile Ile Thr Ile Thr Arg Ile Ile Thr 1 510 Ile Ile Thr Thr Ile Asp Gly Gly Cys Lys Tyr Gly 15 20 Glu Asn Ala ValThr Asn Val Arg Gly Asp Leu Gln 25 30 35 Val Leu Ala Gln Lys Ala Ala ArgCys Leu Pro Thr 40 45 Ser Phe Asn Tyr Gly Ala Ile Lys 50 55 72 aminoacids amino acid linear peptide 102 Thr Ala Lys Ser Lys Lys Phe Pro SerTyr Thr Ala 1 5 10 Thr Tyr Gln Phe Gly Gly Lys Lys Lys Ile Ile Thr 15 20Ile Thr Arg Ile Ile Thr Ile Ile Thr Thr Ile Asp 25 30 35 Gly Gly Cys ThrTyr Gly Thr Gln Pro Ser Arg Arg 40 45 Gly Asp Met Ala Ala Leu Ala GlnArg Leu Ser Arg 50 55 60 Cys Leu Pro Thr Ser Phe Asn Tyr Gly Ala Val Lys65 70 4 amino acids amino acid linear peptide 103 Asn Ala Asn Pro 1 36amino acids amino acid linear peptide 104 Ile Ser Ile Ser Glu Ile LysGly Val Ile Val His 1 5 10 Lys Ile Glu Gly Ile Leu Phe Lys Asn Ala AsnPro 15 20 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 25 30 35 36amino acids amino acid linear peptide 105 Ile Ser Ile Thr Glu Ile ArgThr Val Ile Val Thr 1 5 10 Arg Ile Glu Thr Ile Leu Phe Lys Asn Ala AsnPro 15 20 Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 25 30 35 26amino acids amino acid linear peptide 106 Arg Asp Gly Phe Leu Leu LeuGln Met Asp Phe Gly 1 5 10 Phe Pro Glu His Leu Leu Val Asp Phe Leu GlnSer 15 20 Leu Ser 25 16 amino acids amino acid linear peptide 107 PheGly Phe Pro Glu His Leu Leu Val Asp Phe Leu 1 5 10 Gln Ser Leu Ser 15 16amino acids amino acid linear peptide 108 Phe Gly Phe Pro Lys His LeuLeu Val Asp Phe Leu 1 5 10 Gln Ser Leu Ser 15 26 amino acids amino acidlinear peptide 109 Leu Asp Gly Cys Leu Leu Leu Gln Met Asp Phe Gly 1 510 Phe Pro Lys His Leu Leu Val Asp Phe Leu Gln Ser 15 20 Leu Ser 25 46amino acids amino acid linear peptide 110 Ile Ser Ile Ser Glu Ile LysGly Val Ile Val His 1 5 10 Lys Ile Glu Gly Ile Leu Phe Lys Arg Asp GlyPhe 15 20 Leu Leu Leu Gln Met Asp Phe Gly Phe Pro Glu His 25 30 35 LeuLeu Val Asp Phe Leu Gln Ser Leu Ser 40 45 46 amino acids amino acidlinear peptide 111 Ile Ser Ile Thr Glu Ile Arg Thr Val Ile Val Thr 1 510 Arg Ile Glu Thr Ile Leu Phe Lys Arg Asp Gly Phe 15 20 Leu Leu Leu GlnMet Asp Phe Gly Phe Pro Glu His 25 30 35 Leu Leu Val Asp Phe Leu Gln SerLeu Ser 40 45 36 amino acids amino acid linear peptide 112 Ile Ser IleSer Glu Ile Lys Gly Val Ile Val His 1 5 10 Lys Ile Glu Gly Ile Leu PheLys Phe Gly Phe Pro 15 20 Glu His Leu Leu Val Asp Phe Leu Gln Ser LeuSer 25 30 35 36 amino acids amino acid linear peptide 113 Ile Ser IleThr Glu Ile Arg Thr Val Ile Val Thr 1 5 10 Arg Ile Glu Thr Ile Leu PheLys Phe Gly Phe Pro 15 20 Glu His Leu Leu Val Asp Phe Leu Gln Ser LeuSer 25 30 35 35 amino acids amino acid linear peptide 114 Lys Lys LysIle Ile Thr Ile Thr Arg Ile Ile Thr 1 5 10 Ile Ile Thr Thr Ile Asp LysPhe Gly Phe Pro Glu 15 20 His Leu Leu Val Asp Phe Leu Gln Ser Leu Ser 2530 35 36 amino acids amino acid linear peptide 115 Ile Ser Leu Thr GluIle Arg Thr Val Ile Val Thr 1 5 10 Arg Leu Glu Thr Val Leu Phe Lys PheGly Phe Pro 15 20 Glu His Leu Leu Val Asp Phe Leu Gln Ser Leu Ser 25 3035 36 amino acids amino acid linear peptide 116 Ile Ser Ile Ser Glu IleLys Gly Val Ile Val His 1 5 10 Lys Ile Glu Gly Ile Leu Phe Lys Phe GlyPhe Pro 15 20 Lys His Leu Leu Val Asp Phe Leu Gln Ser Leu Ser 25 30 3536 amino acids amino acid linear peptide 117 Ile Ser Ile Thr Glu Ile ArgThr Val Ile Val Thr 1 5 10 Arg Ile Glu Thr Ile Leu Phe Lys Phe Gly PhePro 15 20 Lys His Leu Leu Val Asp Phe Leu Gln Ser Leu Ser 25 30 35 46amino acids amino acid linear peptide 118 Ile Ser Ile Ser Glu Ile LysGly Val Ile Val His 1 5 10 Lys Ile Glu Gly Ile Leu Phe Lys Leu Asp GlyCys 15 20 Leu Leu Leu Gln Met Asp Phe Gly Phe Pro Lys His 25 30 35 LeuLeu Val Asp Phe Leu Gln Ser Leu Ser 40 45 46 amino acids amino acidlinear peptide 119 Ile Ser Ile Thr Glu Ile Arg Thr Val Ile Val Thr 1 510 Arg Ile Glu Thr Ile Leu Phe Lys Leu Asp Gly Cys 15 20 Leu Leu Leu GlnMet Asp Phe Gly Phe Pro Lys His 25 30 35 Leu Leu Val Asp Phe Leu Gln SerLeu Ser 40 45 35 amino acids amino acid linear peptide 120 Ile Ser IleSer Glu Ile Lys Gly Val Ile Val His 1 5 10 Lys Ile Glu Gly Ile Leu PhePro Pro Xaa Pro Xaa 15 20 Pro Glu His Trp Ser Tyr Gly Leu Arg Pro Gly 2530 35 35 amino acids amino acid linear peptide 121 Ile Ser Ile Thr GluIle Arg Thr Val Ile Val Thr 1 5 10 Arg Ile Glu Thr Ile Leu Phe Pro ProXaa Pro Xaa 15 20 Pro Glu His Trp Ser Tyr Gly Leu Arg Pro Gly 25 30 3534 amino acids amino acid linear peptide 122 Lys Lys Lys Ile Ile Thr IleThr Arg Ile Ile Thr 1 5 10 Ile Ile Thr Thr Ile Asp Pro Pro Xaa Pro XaaPro 15 20 Glu His Trp Ser Tyr Gly Leu Arg Pro Gly 25 30 19 amino acidsamino acid linear peptide 123 Ile Ser Leu Thr Glu Ile Arg Thr Val IleVal Thr 1 5 10 Arg Leu Glu Thr Val Leu Phe 15 19 amino acids amino acidlinear peptide 124 Ile Ser Ile Thr Glu Ile Arg Thr Val Ile Val Thr 1 510 Arg Ile Glu Thr Ile Leu Phe 15 31 amino acids amino acid linearpeptide 125 Glu Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn 1 5 10 AsnThr Arg Lys Ser Ile Arg Ile Gly Pro Gly Gln 15 20 Ala Phe Tyr Ala ThrGly Asp 25 30 51 amino acids amino acid linear peptide Modified site 20/note= “(e-N)Lys” 126 Ile Ser Ile Ser Glu Ile Lys Gly Val Ile Val His 15 10 Lys Ile Glu Gly Ile Leu Phe Xaa Glu Ser Val Glu 15 20 Ile Asn CysThr Arg Pro Asn Asn Asn Thr Arg Lys 25 30 35 Ser Ile Arg Ile Gly Pro GlyGln Ala Phe Tyr Ala 40 45 Thr Gly Asp 50 51 amino acids amino acidlinear peptide Modified site 20 /note= “(e-N)Lys” 127 Ile Ser Ile ThrGlu Ile Arg Thr Val Ile Val Thr 1 5 10 Arg Ile Glu Thr Ile Leu Phe XaaGlu Ser Val Glu 15 20 Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys 2530 35 Ser Ile Arg Ile Gly Pro Gly Gln Ala Phe Tyr Ala 40 45 Thr Gly Asp50 51 amino acids amino acid linear peptide Modified site 20 /note=“(e-N)Lys” 128 Ile Ser Leu Thr Glu Ile Arg Thr Val Ile Val Thr 1 5 10Arg Leu Glu Thr Val Leu Phe Xaa Glu Ser Val Glu 15 20 Ile Asn Cys ThrArg Pro Asn Asn Asn Thr Arg Lys 25 30 35 Ser Ile Arg Ile Gly Pro Gly GlnAla Phe Tyr Ala 40 45 Thr Gly Asp 50 51 amino acids amino acid linearpeptide Modified site 20 /note= “(e-N)Lys” 129 Ile Ser Ile Thr Glu IleArg Thr Val Ile Val Thr 1 5 10 Arg Ile Glu Thr Val Ile Phe Xaa Glu SerVal Glu 15 20 Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys 25 30 35Ser Ile Arg Ile Gly Pro Gly Gln Ala Phe Tyr Ala 40 45 Thr Gly Asp 50 9amino acids amino acid linear peptide 130 Lys Ser Ser Gly Lys Leu IleSer Leu 1 5 9 amino acids amino acid linear peptide 131 Cys Asn Gly ArgLeu Tyr Cys Gly Pro 1 5 10 amino acids amino acid linear peptide 132 CysGly Thr Lys Leu Val Cys Phe Ala Ala 1 5 10 9 amino acids amino acidlinear peptide 133 Lys Arg Ile Val Ile Gly Pro Gln Thr 1 5 9 amino acidsamino acid linear peptide 134 Cys Ala Gly Gly Leu Thr Cys Ser Val 1 5 11amino acids amino acid linear peptide 135 Cys Ser Gly Arg Leu Tyr CysHis Glu Ser Trp 1 5 10 29 amino acids amino acid linear peptide Modifiedsite 10 /note= “(e-N)Lys” 136 Lys Ser Ser Gly Lys Leu Ile Ser Leu XaaIle Ser 1 5 10 Ile Ser Glu Ile Lys Gly Val Ile Val His Lys Ile 15 20 GluGly Ile Leu Phe 25 29 amino acids amino acid linear peptide Modifiedsite 10 /note= “(e-N)Lys” 137 Lys Ser Ser Gly Lys Leu Ile Ser Leu XaaIle Ser 1 5 10 Ile Thr Glu Ile Arg Thr Val Ile Val Thr Arg Ile 15 20 GluThr Ile Leu Phe 25 29 amino acids amino acid linear peptide Modifiedsite 10 /note= “(e-N)Lys” 138 Cys Asn Gly Arg Leu Tyr Cys Gly Pro XaaIle Ser 1 5 10 Ile Ser Glu Ile Lys Gly Val Ile Val His Lys Ile 15 20 GluGly Ile Leu Phe 25 29 amino acids amino acid linear peptide Modifiedsite 10 /note= “(e-N)Lys” 139 Cys Asn Gly Arg Leu Tyr Cys Gly Pro XaaIle Ser 1 5 10 Ile Thr Glu Ile Arg Thr Val Ile Val Thr Arg Ile 15 20 GluThr Ile Leu Phe 25 30 amino acids amino acid linear peptide Modifiedsite 11 /note= “(e-N)Lys” 140 Cys Gly Thr Lys Leu Val Cys Phe Ala AlaXaa Ile 1 5 10 Ser Ile Ser Glu Ile Lys Gly Val Ile Val His Lys 15 20 IleGlu Gly Ile Leu Phe 25 30 30 amino acids amino acid linear peptideModified site 11 /note= “(e-N)Lys” 141 Cys Gly Thr Lys Leu Val Cys PheAla Ala Xaa Ile 1 5 10 Ser Ile Thr Glu Ile Arg Thr Val Ile Val Thr Arg15 20 Ile Glu Thr Ile Leu Phe 25 30 29 amino acids amino acid linearpeptide Modified site 10 /note= “(e-N)Lys” 142 Lys Arg Ile Val Ile GlyPro Gln Thr Xaa Ile Ser 1 5 10 Ile Ser Glu Ile Lys Gly Val Ile Val HisLys Ile 15 20 Glu Gly Ile Leu Phe 25 29 amino acids amino acid linearpeptide Modified site 10 /note= “(e-N)Lys” 143 Lys Arg Ile Val Ile GlyPro Gln Thr Xaa Ile Ser 1 5 10 Ile Thr Glu Ile Arg Thr Val Ile Val ThrArg Ile 15 20 Glu Thr Ile Leu Phe 25 29 amino acids amino acid linearpeptide Modified site 10 /note= “(e-N)Lys” 144 Cys Ala Gly Gly Leu ThrCys Ser Val Xaa Ile Ser 1 5 10 Ile Ser Glu Ile Lys Gly Val Ile Val HisLys Ile 15 20 Glu Gly Ile Leu Phe 25 29 amino acids amino acid linearpeptide Modified site 10 /note= “(e-N)Lys” 145 Cys Ala Gly Gly Leu ThrCys Ser Val Xaa Ile Ser 1 5 10 Ile Thr Glu Ile Arg Thr Val Ile Val ThrArg Ile 15 20 Glu Thr Ile Leu Phe 25 31 amino acids amino acid linearpeptide Modified site 12 /note= “(e-N)Lys” 146 Cys Ser Gly Arg Leu TyrCys His Glu Ser Trp Xaa 1 5 10 Ile Ser Ile Ser Glu Ile Lys Gly Val IleVal His 15 20 Lys Ile Glu Gly Ile Leu Phe 25 30 31 amino acids aminoacid linear peptide Modified site 12 /note= “(e-N)Lys” 147 Cys Ser GlyArg Leu Tyr Cys His Glu Ser Trp Xaa 1 5 10 Ile Ser Ile Thr Glu Ile ArgThr Val Ile Val Thr 15 20 Arg Ile Glu Gly Ile Leu Phe 25 30 52 aminoacids amino acid linear peptide 148 Ile Ser Ile Ser Glu Ile Lys Gly ValIle Val His 1 5 10 Lys Ile Glu Gly Ile Leu Phe Gly Gly Glu Ser Val 15 20Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg 25 30 35 Lys Ser Ile ArgIle Gly Pro Gly Gln Ala Phe Tyr 40 45 Ala Thr Gly Asp 50 52 amino acidsamino acid linear peptide 149 Ile Ser Ile Thr Glu Ile Arg Thr Val IleVal Thr 1 5 10 Arg Ile Glu Thr Ile Leu Phe Gly Gly Glu Ser Val 15 20 GluIle Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg 25 30 35 Lys Ser Ile Arg IleGly Pro Gly Gln Ala Phe Tyr 40 45 Ala Thr Gly Asp 50 50 amino acidsamino acid linear peptide Modified site 20 /note= “(e-N)Lys” 150 Ile SerLeu Thr Glu Ile Arg Thr Val Ile Val Thr 1 5 10 Arg Leu Glu Thr Val LeuPhe Xaa Glu Ser Val Glu 15 20 Ile Asn Cys Thr Arg Pro Asn Asn Asn ThrArg Lys 25 30 35 Ser Ile Arg Ile Gly Pro Gly Gln Ala Phe Tyr Ala 40 45Thr Gly 50 50 amino acids amino acid linear peptide Modified site 20/note= “(e-N)Lys” 151 Ile Ser Ile Thr Glu Ile Arg Thr Val Ile Val Thr 15 10 Arg Ile Glu Thr Val Ile Phe Xaa Glu Ser Val Glu 15 20 Ile Asn CysThr Arg Pro Asn Asn Asn Thr Arg Lys 25 30 35 Ser Ile Arg Ile Gly Pro GlyGln Ala Phe Tyr Ala 40 45 Thr Gly 50

I claim:
 1. A T helper cell epitope selected from the group consistingof SEQ ID NO: 6-22, 105, 123, 124, and 31-35.
 2. A T helper cell epitopeaccording to claim 1 for preparing a peptide immunogen represented bythe formula (A)_(n)-(Targent antigentic site)-(B)_(o)-(Th)_(m)-X or(A)_(n)-(Th)_(m)-(B)_(o)-(Target antigenic site)-X or(A)_(n)-(B)_(o)(Th)_(m)-(B)_(o)-(Target antigenic site)-X or Targentantigenic site)-(B)_(o)-(Th)_(m)-(A)_(n)-X or (Th)_(m)-(B)_(o)-(Targetantigenic site)-(A)_(n)-X wherein A is an amino acid or a generalimmunostimulatory sequence, where n is more than one, the interval A'smay be the same or different; B selected from the group consisting ofamino acids, —HCH(X)CH₂SCH₂CO—, NHCH(X)CH₂SCH₂CO(—N)Lys—,—NHCH(X)CH₂S-succinimidyl(□-N)Lys, and NHCH(X)CH₂S-(succinimidyl); Th isan artificial helper T cell epitope selected from the group of SEQ IDNOS:6-22, 105, 31-35 and an analog thereof: “Target antigenic site” isselected from the group consisting of a B cell epitope, a peptidehapten, and a immunologically reactive anaolog thereof; X is amino acidα-COOH or CONH₂, n is from 1 to about 10; m is from 1 to about 4; and ois from 0 to about
 10. 3. A peptide immunogen according to claim 2wherein the immunostimulatory sequence is SEQ ID NO:78.
 4. A peptideimmunogen according to claim 2 wherein B is selected from the groupconsisting of Gly—Gly, (□-N)Lys, Pro-Pro-Xaa-Pro-Pro,—NHCH(X)CH₂SCH₂CO—, —NHCH(X)CH₂SCH₂CO (□-N)Lys-,—NHCH(X)CH₂S-succinimidyl (□-N)Lys-, and —NHCH(X)CH₂S-(succinimidly)-.5. A peptide immunogen according to claim 4 wherein B is Gly—Gly.
 6. Apeptide immunogen according to claim 4 wherein B is (□-N)Lys.
 7. Apeptide immunogen according to claim 1, 2, 3, 4, 5, or 6 wherein the theTarget Antigen Site is the Plasmodium falciparum repeating antigen:(Asn-Ala-Asn-Pro)_(p) (SEQ ID NO:103).
 8. A peptide immunogen accordingto claim 7 wherein p=4.
 9. A peptide immunogen according to claim 7selected from the group consisting of SEQ ID NOs: 104, and
 105. 10. Apeptide immunogen according to claim 1, 2, 3, 4, 5, or 6 wherein the theTarget Antigen site is selected from the group consisting of SEQ ID NO:106, 107, 108, and 109, an epitope of CETP.
 11. A peptide immunogenaccording to claim 10 selected from the group consisting of SEQ IDNOs:110-118, and
 119. 12. A peptide immunogen according to claim 1, 2,3, 4, 5, or 6 wherein the the Target Antigen site is selected from thegroup consisting of SEQ ID NOS: 125, 131, 132, 133, 134, and 135, andepitope of HIV.
 13. A peptide immunogen according to claim 12 selectedfrom the group consisting of SEQ ID NOs:126-129, and 136-151.
 14. Apeptide immunogen according to claim 13 selected from the groupconsisting of SEQ ID NOs:148-150, and
 151. 15. A method for producing apeptide immunogen by covalently linking a T helper cell epitope to atarget antigenic site selected from the group consisting of B cellepitopes of an antigen and a peptide hapten.
 16. A method for producinga peptide immunogen according to claim 15 further linking the covalentlylinked T helper cell epitope and target antigenic site to animmunostimulatory sequence.
 17. A method for producing a peptideimmunogen according to claim 16 wherein the immunostimulatory sequenceis SEQ ID NO:78.
 18. A method for producing a peptide immunogenaccording to claim 17 wherein B is selected from the group consisting ofGly—Gly, (Δ-N)Lys, Pro-Pro-Xaa-Pro-Pro, —NHCH(X)CH₂SCH₂CO—,—NHCH(X)CH₂SCH₂CO (□-N)Lys-, —NHCH(X)CH₂S-succinimidyl (□-N)Lys-, and—NHCH(X)CH₂S-succinimidyl)-.
 19. A method for producing a peptideimmunogen according to claim 18 wherein B is Gly—Gly.
 20. A method forproducing a peptide immunogen according to claim 19 wherein B is(□-N)Lys.
 21. A method of inducing T helper cell response by employing apeptide immunogen of claim
 1. 22. A method of inducing T helper cellresponse by employing a peptide immunogen of of claim
 2. 23. A method ofinducing T helper cell response to employing a peptide immunogen of ofclaim
 3. 24. A method of inducing T helper cell response by employing apeptide immunogen of of claim
 4. 25. A method of inducing T helper cellresponse by employing a peptide immunogen of of claim
 5. 26. A method ofinducing T helper cell response by employing a peptide immunogen of ofclaim
 6. 27. A method of inducing T helper cell response by employing apeptide immunogen of of claim
 7. 28. A method of inducing T helper cellresponse by employing a peptide immunogen of of claim
 8. 29. A method ofinducing T helper cell response by employing a peptide immunogen of ofclaim
 9. 30. A method of inducing T helper cell response by employing apeptide immunogen of of claim
 10. 31. A method of inducing T helper cellresponse by employing a peptide immunogen of of claim
 11. 32. A methodof inducing T helper cell response by employing a peptide immunogen ofof claim
 12. 33. A method of inducing T helper cell response byemploying a peptide immunogen of of claim 13.